Avirulent, immunogenic flavivirus chimeras

ABSTRACT

Chimeric flaviviruses that are avirulent and immunogenic are provided. The chimeric viruses are constructed to contain amino acid mutations in the nonstructural viral proteins of a flavivirus. Chimeric viruses containing the attenuation-mutated nonstructural genes of the virus are used as a backbone into which the structural genes of a second flavivirus strain are inserted. These chimeric viruses elicit pronounced immunogenicity yet lack the accompanying clinical symptoms of viral disease. The attenuated chimeric viruses are effective as immunogens or vaccines and may be combined in a pharmaceutical composition to confer simultaneous immunity against several strains of pathogenic flaviviruses.

This is a divisional of U.S. patent application Ser. No. 11/506,251,filed Aug. 18, 2006, now pending; which is a divisional of U.S. patentapplication Ser. No. 10/204,252, filed Aug. 16, 2002, now U.S. Pat. No.7,094,411, issued Aug. 22, 2006; which is the 35 U.S.C. §371 nationalphase of international application PCT/US01/05142, filed Feb. 16, 2001(published under PCT Article 21(2) in English); and which claims thebenefit of provisional application Ser. No. 60/182,829, filed Feb. 16,2000. Each of these applications is herein incorporated by reference.

This invention was made by the Centers for Disease Control andPrevention, an agency of the United States Government. Therefore, theUnited States Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the fields of immunology and virologyand more particularly to avirulent, immunogenic flavivirus chimeras forthe production of immunogenic, live, attenuated flavivirus vaccines.

BACKGROUND OF THE INVENTION

Dengue viruses are mosquito-borne pathogens of the genes Flavivirus(family Flaviviridae). Four serotypes of dengue viruses (oftenabbreviated “DEN”) have been identified, including dengue-1, dengue-2,dengue-3 and dengue-4 (DEN-1 to DEN-4). The flavivirus genome is asingle-stranded, positive-sense RNA approximately 11 kb in length,containing a 5′-noncoding region (5′NC); a coding region encoding theviral structural proteins; five nonstructural proteins, designated NS1,NS2A, NS2B, NS3, NS4A, NS4B, NS5; and a 3′-noncoding region (3′NC). Theviral structural proteins include the capsid, premembrane/membrane, andenvelope. The structural and nonstructural proteins are translated as asingle polyprotein. The polyprotein is then processed by cellular andviral proteases.

Transmitted by Aedes aegypti mosquitoes to humans in tropical andsubtropical regions of the world, dengue viruses cause millions of casesof disease every year, ranging from dengue fever to the often fataldengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Secondaryinfection of humans with a heterologous serotype of DEN virus may inducean immunopathological response and is considered a possible risk factorfor DHF/DSS. Therefore, the need exists for development of a vaccinethat confers simultaneous protection against all dengue virus strains.

Since eradication of Aedes aegypti mosquitoes appears to be practicallyinfeasible, development of safe, effective vaccines against all fourserotypes of dengue virus is a World Health Organization priority.However, no approved, effective vaccine against any of the dengue virusstrains is currently available. It has been demonstrated that serialpassage of wild-type flaviviruses in various cell cultures, such asprimary dog kidney (PDK) cells, produces virus variants that havereduced virulence, retain immunogenicity and produce no untowardclinical symptoms.

Live, attenuated dengue viruses of all four serotypes have beendeveloped at Mabidol University in Thailand by passaging the wild-typeviruses in cell culture. These are currently the most promising live,attenuated vaccine candidates for immunization against dengue virusinfection and/or disease. These vaccine candidates have been designatedby a combination of their dengue serotype, the cell line through whichthey were passaged and the number of times they were passaged. Thus, adengue serotype 1 wild-type virus passaged in PDK cells 13 times isdesignated as DEN-1 PDK-13 virus (nucleotide sequence, SEQ ID NO:3;amino acid sequence, SEQ ID NO:4). The other vaccine candidates areDEN-2 PDK-53 (nucleotide sequence, SEQ ID NO:15; amino acid sequence,SEQ ID NO:16), DEN-3 PGMK-30/FRhL-3 (thirty passages in primary greenmonkey kidney cells, followed by three passages in fetal rhesus lungcells)(nucleotide sequence, SEQ ID NO:21; amino acid sequence, SEQ IDNO:22) and DEN-4 PDK-48 (nucleotide sequence, SEQ ID NO:25; amino acidsequence, SEQ ID NO:26). These four candidate vaccine viruses werederived by tissue culture passage of wild-type parental DEN-1 16007(nucleotide sequence, SEQ ID NO:1; amino acid sequence, SEQ ID NO:2),DEN-2 16681(nucleotide sequence, SEQ ID NO:13; amino acid sequence, SEQID NO:14), DEN-3 16562 (nucleotide sequence, SEQ ID NO:19; amino acidsequence, SEQ ID NO:20) and DEN-4 1036 (nucleotide sequence, SEQ IDNO:23; amino acid sequence, SEQ ID NO:24) viruses, respectively.

Preliminary human clinical trials with these attenuated viruses haveindicated that DEN-2 PDK-53 has the lowest infectious dose (50% minimalinfectious dose of 5 plaque forming units or PFU) in humans, is stronglyimmunogenic, and produces no unacceptable clinical symptoms. The DEN-1PDK-13, DEN-3 PGMK-30/FRhL-3 and DEN-4 PDK-48 vaccine virus candidateshave higher 50% minimal infectious doses of 10,000, 3500, and 150 PFU,respectively, in humans. The higher infectious doses required for thelatter three vaccine candidates raises concerns regarding the relativeefficacy of each serotype component in a tetravalent dengue virusvaccine. Although only one immunization with monovalent DEN-2 PDK-53virus or DEN-4 PDK-48 virus was required to achieve 100% seroconversionin human subjects, a booster was needed to achieve the sameseroconversion rate for DEN-1 PDK-13 and DEN-3 PGMK-30/FRhL-3 viruses,which have the two highest infectious doses for humans.

The DEN-2 PDK-53 virus vaccine candidate, henceforth abbreviated PDK-53,has several measurable biological markers associated with attenuation,including temperature sensitivity, small plaque size, decreasedreplication in mosquito C6/36 cell culture, decreased replication inintact mosquitoes, loss of neurovirulence for suckling mice anddecreased incidence of viremia in monkeys. Clinical trials of thecandidate PDK-53 vaccine have demonstrated its safety and immunogenicityin humans. Furthermore, the PDK-53 vaccine induces dengue virus-specificT-cell memory responses in human vaccine recipients.

Except for DEN-2 PDK-53 virus, the number and identity of the geneticmutations that accrued during multiple passages in cell culture and thatare associated with the attenuated phenotypes of the vaccine candidatesare unknown. Neither the relative contributions of suchattenuation-associated mutations to the actual mechanism of attenuation,nor the potential for reverse mutations to revert any of the vaccinecandidates to the virulent biological phenotype of the wild-type denguevirus are known for any of these four vaccine candidates. Anunderstanding of the attenuation markers of a vaccine candidate iscritical for the prediction of its stability and safety.

Accordingly, there is a need for avirulent, yet immunogenic, dengueviruses to be used in the development of dengue virus vaccines to conferprotection against one or more dengue virus serotypes. What would beideal is a vaccine that would simultaneously protect an individualagainst several virulent strains of this potentially dangerous family(Flaviviridae) of viruses. Therefore, a tetravalent vaccine that can beused to immunize an individual against all four dengue serotypes isparticularly needed.

SUMMARY OF THE INVENTION

Immunogenic flavivirus chimeras, a dengue-2 virus backbone for preparingthe flavivirus chimeras and methods for producing the flaviviruschimeras are described. The immunogenic flavivirus chimeras areprovided, alone or in combination, in a pharmaceutically acceptablecarrier as immunogenic compositions to minimize, inhibit, or immunizeindividuals against infection by one or more flaviviruses or flaviviralstrains, particularly strains of the dengue virus serotypes DEN-1,DEN-2, DEN-3 and DEN-4. When combined, the immunogenic flaviviruschimeras may be used as multivalent vaccines to confer simultaneousprotection against infection by more than one species or strain offlavivirus. Preferably, the flavivirus chimeras are combined in animmunogenic composition useful as a tetravalent vaccine against the fourknown dengue virus serotypes. The nucleic acid sequence for each of theDEN-1, DEN-3 and DEN-4 viruses is also provided, for use as probes todetect dengue virus in a biological sample.

The avirulent, immunogenic flavivirus chimeras provided herein containthe nonstructural protein genes of the attenuated dengue-2 virus, or theequivalent thereof, and one or more of the structural protein genes orimmunogenic portions thereof of the flavivirus against whichimmunogenicity is to be conferred. For example, the preferred chimeracontains the attenuated dengue-2 virus PDK-53 genome as the viralbackbone, and the structural protein genes encoding capsid,premembrane/membrane, or envelope of the PDK-53 genome, or combinationsthereof, are replaced with the corresponding structural protein genesfrom a flavivirus to be protected against, such as a differentflavivirus or a different dengue virus serotype. The resulting viralchimera has the functional properties of the attenuated dengue-2 virusand is therefore avirulent, but expresses antigenic epitopes of thestructural gene products and is therefore immunogenic.

In another embodiment, the preferred chimera is a nucleic acid chimeracomprising a first nucleotide sequence encoding nonstructural proteinsfrom an attenuated dengue-2 virus, and a second nucleotide sequenceencoding a structural protein from a second flavivirus. In a furtherpreferred embodiment, the attenuated dengue-2 virus is vaccine strainPDK-53. In a further preferred embodiment, the structural protein can bethe C, prM or E protein of a flavivirus. Examples of flaviviruses fromwhich the structural protein may be selected include, but are notlimited to, dengue-1 virus, dengue-2 virus, dengue-3 virus, dengue-4virus, West Nile virus, Japanese encephalitis virus, St. Louisencephalitis virus, yellow fever virus and tick-borne encephalitisvirus. In a further embodiment, the structural protein may be selectedfrom non-flavivirus species that are closely related to theflaviviruses, such as hepatitis C virus.

In evaluating the chimeric virus of the invention, it was unexpectedlydiscovered that the avirulence of the attenuated PDK-53 virus strain isattributable to the presence of specific amino acid substitutionmutations in the nonstructural proteins and a nucleotide substitutionmutation in the 5′ noncoding region. This nucleotide substitutionmutation occurs in the stem of a stem-loop structure that is conservedin all four dengue serotypes. In particular, a single mutation atNS1-53, a double mutation at NS1-53 and at 5′NC-57, a double mutation atNS1-53 and at NS3-250, and a triple mutation at NS1-53, at 5′NC-57 andat NS3-250, can provide the attenuated DEN-2 virus of the presentinvention.

Furthermore, the genome of any dengue-2 virus containingnon-conservative amino acid substitutions at these loci can be used asthe backbone in the avirulent chimeras described herein. Furthermore,other flavivirus genomes containing analogous mutations at the sameloci, after amino acid sequence or nucleotide sequence alignment andstem structure analysis can also be used as the backbone structure andare defined herein as being equivalent to attenuating mutations of thedengue-2 PDK-53 genome.

The backbone, that region of the chimera that comprises the 5′ and 3′noncoding regions and the region encoding the nonstructural proteins,can also contain further mutations to maintain stability of theavirulent phenotype and to reduce the possibility that the avirulentvirus or chimera might revert back to the virulent wild-type virus. Forexample, a second mutation in the stem of the stem/loop structure in the5′ noncoding region will provide additional stability, if desired.

These chimeric viruses can comprise nucleotide and amino acidsubstitutions, deletions or insertions in their structural andnonstructural proteins in addition to those specifically describedherein.

The structural and nonstructural proteins of the invention are to beunderstood to include any protein comprising or any gene encoding thesequence of the complete protein, an epitope of the protein, or anyfragment comprising, for example, two or more amino acid residuesthereof.

The present invention also provides a method for making the chimericviruses of this invention using recombinant techniques, by inserting therequired substitutions into the appropriate backbone genome.

The present invention also provides compositions comprising apharmaceutically acceptable carrier and attenuated chimeric viruses ofthis invention which contain amino acid sequences derived from otherdengue virus serotypes, other flavivirus species or other closelyrelated species, such as hepatitis C virus. As an object of theinvention, the amino acid sequences derived from other dengue virusserotypes, other flavivirus species or other closely related species,such as hepatitis C virus, are expressed in a host, host cell or cellculture. As a further object of the invention, proteins or polypeptidescomprising the amino acid sequences derived from other dengue virusserotypes, other flavivirus species or other closely-related species,can act as immunogens and, thus, be used to induce an immunogenicresponse against other dengue virus serotypes, other flavivirus speciesor other closely related species.

The present invention also provides compositions comprising apharmaceutically acceptable carrier, one or more attenuated chimericviruses of this invention and further immunizing compositions. Examplesof such further immunizing compositions include, but are not be limitedto, dengue virus vaccines, yellow fever virus vaccines, tick-borneencephalitis virus vaccines, Japanese encephalitis virus vaccines, WestNile virus vaccines, hepatitis C virus vaccines or other virus vaccines.Such vaccines may be live attenuated virus vaccines, killed virusvaccines, subunit vaccines, recombinant DNA vector vaccines or anycombination thereof.

A distinct advantage of the current invention is that it provides formixtures of attenuated flavivirus chimeras to be used as vaccines inorder to impart immunity against several flavivirus speciessimultaneously.

Thus, an object of the current invention is to provide a virus chimeracontaining amino acid or nucleotide substitutions which retainimmunogenicity of the virus while preventing any pathogenic effects ofthe virus.

Another object of the present invention is to provide nucleic acidchimeras comprising nucleotide sequence from an attenuated dengue-2virus and nucleotide sequence from a second flavivirus, wherein thenucleotide sequence from the second flavivirus directs the synthesis offlavivirus antigens.

Another object of the present invention is to provide compositions forvaccines comprising more than one flavivirus species.

Another object of the present invention is to provide a method formaking immunogenic or vaccine compositions using recombinant techniquesby inserting the required substitutions into an appropriate flavivirusgenome.

Another object of the invention is to provide compositions and methodsfor imparting immunity against more than one flavivirus simultaneously.

Another object of the invention is to provide nucleic acid probes andprimers for use in any of a number of rapid genetic tests that arediagnostic for each of the vaccine viruses of the current invention.This object of the invention may be embodied in polymerase chainreaction assays, hybridization assays or other nucleic acid sequencedetection techniques known to the art. A particular embodiment of thisobject is an automated PCR-based nucleic acid detection system.

These and other objects, features and advantages of the presentinvention will become apparent after review of the following detaileddescription of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically shows the genomic organization of the chimericDEN-2/DEN-1 viruses. Designations of the chimeras are based on the DEN-2virus-specific infectious clone backbones and the structural genes(C-prM-E) insert of DEN-1 viruses. Underlined letters of the backboneand insert viruses are used in the designations. D2/I indicatesDEN-2/DEN-1 chimera; the first letter following the hyphen is the DEN-2viral backbone, parent 16681 (P), PDK53-E (E), or PDK53-V (V); the lastletter indicates the structural genes from the parental DEN-1 16007 (P)strain or its vaccine derivative, strain PDK-13 (V). The PDK53-Ebackbone contains three DEN-2 PDK-53 virus-specific amino acid mutations(NS1-53-Asp, NS2A-181-Phe, and NS4A-75-Ala) as well as the 5′NC-57mutation of PDK-53 virus. The PDK53-V backbone contains these samePDK-53 virus-specific loci plus the additional PDK-53 virus-specificNS3-250-Val locus.

FIGS. 2A and 2B show the growth characteristics of the chimericDEN-2/DEN-1 viruses in LLC-MK₂ cells. Stippled bars indicate DEN-1 16007virus and the chimeric viruses expressing the structural proteins ofDEN-1 16007 virus. Bars with stripes indicate DEN-1 PDK-13 virus and thechimeric viruses expressing structural proteins of PDK-13 virus. Blankbars indicate the three DEN-2 backbone viruses derived from infectiousclones of DEN-2 16681 virus (P48) and the two variants (PDK53-E andPDK53-V; E48 and V48, respectively). FIG. 2A: Mean(±SD) plaquediameters. Values were calculated from ten individual plaques of eachvirus on day 10 after infection. pp: pinpoint-size plaques less than 1mm. FIG. 2B: Temperature sensitivity (ts) and peak titers of chimericviruses on day 8 or 10 after infection. The ts scores were based on thereduction of the virus titers at 38.7° C. versus those at 37° C. (−, +,2+ and 3+ indicate titer reduction of less than or equal to 60%, 61-90%,91-99%, >99%, respectively, calculated from at least three experiments).The graph bar heights represent the log₁₀ titers of the viruses at 37°C. The multiplicity of infection (m.o.i.) was approximately 0.001PFU/cell.

FIG. 3 shows the growth curves of DEN-1 16007, DEN-1 PDK-13, DEN-216681-P48, DEN-2 PDK53-E48, DEN-2 PDK53-V48 and chimeric DEN-2/DEN-1viruses in C6/36 cells. Cells were infected at an approximate m.o.i. of0.001 PFU/ml.

FIGS. 4A-C. FIG. 4A: Mean plaque diameters±SD in millimeters (n=12) ofDEN-2 16681, PDK-53 and recombinant 166811PDK-53 viruses at nine daysafter infection in LLC-MK₂ cells. FIG. 4B: Peak replication titers at6-8 days after infection of LLC-MK₂ cells at a m.o.i. of approximately0.001 PFU/cell in a single experiment. Temperature sensitivity (ts)scores for viruses grown at 37° C. or 38.7° C. in LLC-MK₂ cells areshown above the graph bars for peak replication titers. Scores of (−),(+/−) and (+) indicate less than 81%, 81-89% and 90-97% reduction inviral titer, respectively, at 38.7° C. Scores were determined at eightdays after infection. FIG. 4C: Average peak replication titers at 12days after infection of C6/36 cells at a multiplicity of approximately0.001 PFU/cell in two independent experiments. Individual peak titersfrom the two experiments are indicated by vertical lines in each graphbar. The numerical designations for recombinant Px and Vx viruses (wherex=5′NC, NS1, and/or NS3 loci) indicate parental (P in virus designation)16681 virus-specific loci engineered into the PDK-53 virus-specificinfectious cDNA clone or reciprocal candidate PDK-53 vaccine (V in virusdesignation) virus-specific loci engineered into the 16681 clone,respectively. Cognate viruses are indicated in all three graphs by graphbars of identical solid or cross-hatching pattern. The cognate for P5virus is V13 virus, assuming that the viral phenotype is determinedpredominantly by the 5′NC-57, NS1-53 and NS3-250 loci. Both P5 and V13viruses contain the 5′NC-57-C (16681), NS1-53-Asp (PDK-53) andNS3-250-Val (PDK-53) loci within the genetic backgrounds of PDK-53 and16681 viruses, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The following description includes the best presently contemplated modeof carrying out the invention. This description is made for the purposeof illustrating the general principles of the inventions and should notbe taken in a limiting sense.

Immunogenic flavivirus chimeras, a dengue-2 virus or dengue-2 virusequivalent backbone for preparing the flavivirus chimeras of thisinvention and methods for preparing the flavivirus chimeras are providedherein. The immunogenic flavivirus chimeras are useful, alone or incombination, in a pharmaceutically acceptable carrier as immunogeniccompositions to minimize, inhibit, or immunize individuals againstinfection by one or more flaviviruses or flaviviral strains,particularly strains of the dengue virus serotypes DEN-1, DEN-2, DEN-3and DEN-4. When combined, the immunogenic flavivirus chimeras may beused as a multivalent vaccine to confer simultaneous protection againstinfection. Preferably, the dengue virus chimeras are combined in animmunogenic composition useful as a tetravalent vaccine against the fourknown dengue virus serotypes.

Immunogenic flavivirus chimeras of the current invention are alsouseful, in combination with avirulent virus strains, in apharmaceutically acceptable carrier, as immunogenic compositions tominimize, inhibit or immunize individuals against infection by multiplepathogenic species. For example, one or more of the immunogenicflavivirus chimeras of the current invention can be combined withavirulent virus serotypes of selected flaviviruses to provide a safe andeffective tetravalent vaccine against the four known dengue virusserotypes. In a further embodiment, the flavivirus chimeras of thecurrent invention may be combined with avirulent virus vaccines toprovide a safe and effective vaccine against infection by multiplepathogenic species.

The present invention also provides compositions comprising apharmaceutically acceptable carrier, one or more attenuated chimericviruses of this invention and further immunizing compositions. Examplesof such further immunizing compositions include, but are not be limitedto, dengue virus vaccines, yellow fever vaccines, tick-borneencephalitis vaccines, Japanese encephalitis vaccines, West Nile virusvaccines, hepatitis C virus vaccines or other virus vaccines. Suchvaccines may be live attenuated virus vaccines, killed virus vaccines,subunit vaccines, recombinant DNA vector vaccines or any combinationthereof.

The nucleic acid sequence for each of the DEN-1, DEN-3 and DEN-4 virusesis also provided for use as probes to detect dengue virus in abiological sample.

Chimeras of the present invention can comprise the backbone of thedengue-2 virus of an attenuated dengue-2 virus and further nucleotidesequences selected from more than one dengue virus serotype, otherflavivirus species, other closely related species, such as hepatitis Cvirus, or any combination thereof. These chimeras can be used to inducean immunogenic response against more than one species selected from thedengue virus serotypes, flavivirus species, other closely relatedspecies or any combination thereof.

In another embodiment, the preferred chimera is a nucleic acid chimeracomprising a first nucleotide sequence encoding nonstructural proteinsfrom an attenuated dengue-2 virus, and a second nucleotide sequenceencoding a structural protein from a second flavivirus. In a furtherpreferred embodiment, the attenuated dengue-2 virus is vaccine strainPDK-53. In a further preferred embodiment, the structural protein can bethe C protein of a flavivirus, the prM protein of a flavivirus, the Eprotein of a flavivirus, or any combination thereof. Examples offlaviviruses from which the structural protein may be selected include,but are not limited to, dengue-1 virus, dengue-2 virus, dengue-3 virus,dengue-4 virus, West Nile virus, Japanese encephalitis virus, St. Louisencephalitis virus, yellow fever virus and tick-borne encephalitisvirus. In a further embodiment, the structural protein may be selectedfrom non-flavivirus species that are closely related to theflaviviruses, such as hepatitis C virus.

The terms “a,” “an” and “the” as used herein are defined to mean one ormore and include the plural unless the context is inappropriate.

The term “residue” is used herein to refer to an amino acid (D or L) oran amino acid mimetic that is incorporated into a peptide by an amidebond. As such, the amino acid may be a naturally occurring amino acidor, unless otherwise limited, may encompass known analogs of naturalamino acids that function in a manner similar to the naturally occurringamino acids (i.e., amino acid mimetics). Moreover, an amide bond mimeticincludes peptide backbone modifications well known to those skilled inthe art.

Furthermore, one of skill in the art will recognize that individualsubstitutions, deletions or additions in the amino acid sequence, or inthe nucleotide sequence encoding for the amino acids, which alter, addor delete a single amino acid or a small percentage of amino acids(typically less than 5%, more typically less than 1%) in an encodedsequence are conservatively modified variations, wherein the alterationsresult in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

As used herein, the terms “virus chimera,” “chimeric virus,” “flaviviruschimera” and “chimeric flavivirus” means an infectious construct of theinvention comprising a portion of the nucleotide sequence of a dengue-2virus and further nucleotide sequence that is not from the same dengue-2virus. Thus, examples of further nucleotide sequence include, but arenot limited to, sequences from dengue-1 virus, dengue-2 virus, dengue-3virus, dengue-4 virus, West Nile virus, Japanese encephalitis virus, St.Louis encephalitis virus, tick-borne encephalitis virus, yellow fevervirus and any combination thereof.

As used herein, “infectious construct” indicates a virus, a viralconstruct, a viral chimera, a nucleic acid derived from a virus or anyportion thereof, which may be used to infect a cell.

As used herein, “nucleic acid chimera” means a construct of theinvention comprising nucleic acid comprising a portion of the nucleotidesequence of a dengue-2 virus and further nucleotide sequence that is notof the same origin as the nucleotide sequence of the dengue-2 virus.Correspondingly, any chimeric flavivirus or flavivirus chimera of theinvention is to be recognized as an example of a nucleic acid chimera.

The structural and nonstructural proteins of the invention are to beunderstood to include any protein comprising or any gene encoding thesequence of the complete protein, an epitope of the protein, or anyfragment comprising, for example, two or more amino acid residuesthereof.

Nucleotide sequences of the RNA genome of the viruses and chimeras ofthe current invention are recited in the sequence listings in terms ofDNA.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one having ordinaryskill in the art to which this invention pertains. Although othermaterials and methods similar or equivalent to those described hereincan be used in the practice or testing of the present invention, thepreferred methods and materials are now described.

Flavivirus Chimeras

Dengue virus types 1-4 (DEN-1 to DEN-4) are mosquito-borne flaviviruspathogens. The flavivirus genome contains a 5′-noncoding region (5′-NC),followed by a capsid protein (C) encoding region, followed by apremembrane/membrane protein (prM) encoding region, followed by anenvelope protein (E) encoding region, followed by the region encodingthe nonstructural proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5) and finallya 3′ noncoding region (3′NC). The viral structural proteins are C, prMand E, and the nonstructural proteins are NS1-NS5. The structural andnonstructural proteins are translated as a single polyprotein andprocessed by cellular and viral proteases.

The flavivirus chimeras of the invention are constructs formed by fusingnon-structural protein genes from one type, or serotype, of dengue virusor virus species of the flaviviridae, with protein genes, for example,structural protein genes, from a different type, or serotype, of denguevirus or virus species of the flaviviridae. Alternatively, a flaviviruschimera of the invention is a construct formed by fusing non-structuralprotein genes from one type, or serotype, of dengue virus or virusspecies of the flaviviridae, with further nucleotide sequences thatdirect the synthesis of polypeptides or proteins selected from otherdengue virus serotypes or other viruses of the flaviviridae.

The avirulent, immunogenic flavivirus chimeras provided herein containthe nonstructural protein genes of the attenuated dengue-2 virus, or theequivalent thereof and one or more of the structural protein genes, orantigenic portions thereof, of the flavivirus against whichimmunogenicity is to be conferred. Suitable flaviviruses include, butare not limited to those listed in Table 1.

Other suitable flaviviruses for use in constructing the flaviviruschimeras of the invention are wild-type, virulent DEN-1 16007 (SEQ IDNO:1; SEQ ID NO:2), DEN-2 16681 (SEQ ID NO:13; SEQ ID NO:14), DEN-316562 (SEQ ID NO:19; SEQ ID NO:20) and DEN-4 1036 (SEQ ID NO:23; SEQ IDNO:24) and attenuated, vaccine-strain DEN-1 PDR-13 (SEQ ID NO:3; SEQ IDNO:4), DEN-2 PDK-53 (SEQ ID NO:15; SEQ ID NO:16), DEN-3 PMK-30/FRhL-3(SEQ ID NO:21; SEQ ID NO:22) and DEN-4 PDK-48 (SEQ ID NO:25; SEQ IDNO:26). Further suitable flaviviruses, or variants of the above listedflaviviruses, are described herein. Genetic differences between theDEN-1, DEN-2, DEN-3 and DEN-4 wild type/attenuated virus pairs are shownin Tables 2-5 along with changes in the amino acid sequences encoded bythe viral genomes.

The sequence listings for DEN-2 PDK-53 provided herein (SEQ ID NO:15;SEQ ID NO:16) correspond to the DEN-2 PDK-53-V variant, wherein genomenucleotide position 5270 is mutated from an A to a T and amino acidposition 1725 of the polyprotein or amino acid position 250 of the NS3protein contains a valine residue. The DEN-2 PDK-53 variant without thisnucleotide mutation, DEN-2 PDK-53-E, differs from PDK-53-V only in thisone position. DEN-2 PDK-53-E has an A at nucleotide position 5270 and aglutamate at polyprotein amino acid position 1725, NS3 protein aminoacid position 250 (Table 3).

The sequence listings for DEN-3 16562 provided herein (SEQ ID NO:21; SEQID NO:22) correspond to the variant wherein genome nucleotide position1521 is a T and amino acid position 476 of the polyprotein or amino acidposition 196 of the E protein contain a leucine. A second variant,present in DEN-3 16562 cultures has a T at nucleotide position 1521 andamino acid position 476 of the polyprotein or amino acid position 196 ofthe E protein contain a serine (Table 4).

The sequence listings for DEN-4 PDK-48 (SEQ ID NO:25; SEQ ID NO:26)correspond to the variant wherein genome nucleotide positions: 6957 is aT and amino acid position 2286 of the polyprotein and amino acidposition 44 of NS4B protein is a phenylalanine, 7546 is a T and aminoacid position 2366 of the polyprotein and amino acid position 240 ofNS4B protein is a valine, and 7623 is a T and amino acid position 2508of the polyprotein and amino acid position 21 of NS5 protein is atyrosine (Table 5).

Throughout the text, designations of the chimeras are based on the DEN-2virus-specific infectious clone backbones and the structural genes(prM-E or C-prM-E) insert of other flaviviruses. Each designation beginswith DEN-2 for the dengue-2 backbone, followed by the strain from whichthe structural genes are inserted. The particular backbone variant isreflected in the next letter. The particular DEN-2 backbone variant fromwhich the chimera was constructed is indicated by the following letterplaced after a hyphen, parent 16681 (P), PDK53-E (E), or PDK53-V (V);the last letter indicates the C-prM-E structural genes from the parental(P) strain or its vaccine derivative (V) or the prM-E structural genesfrom the parental (P1) or its vaccine derivative (V1). For example;DEN-2/1-VP (SEQ ID NO:5; SEQ ID NO:6) denotes the chimera comprising theattenuated DEN-2 PDK-53V backbone comprising a valine at NS3-250 and theC-prM-E genes from wild-type DEN-1 16007; DEN-2,1-VV (SEQ ID NO:7; SEQID NO:8) denotes the DEN-2 PDK-53V backbone with the vaccine strain ofdengue-1, DEN-1 PDK-13; DEN-2/1-VP1 (SEQ ID NO:27; SEQ ID NO:28) denotesthe DEN-2 PDK-53V backbone and the prM-E genes from wild-type DEN-116007; DEN-2/3-VP1 (SEQ ID NO:9; SEQ ID NO:10) denotes the DEN-2 PDK-53Vbackbone and the prM-E genes from wild-type DEN-3 16562; DEN-2/4-VP1(SEQ ID NO:11; SEQ ID NO:12) denotes the DEN-2 PDK-53V backbone and theprM-E genes from wild-type DEN-4 1036; and DEN-2/WN-PP1 (SEQ ID NO:17;SEQ ID NO:18) denotes the DEN-2 16681 backbone and the prM-E genes fromWest Nile NY99. Other chimeras of the present invention, denoted in thesame manner, are clearly defined herein by the disclosed sequences. Forinstance, DEN-2/1-PV is defined herein as consisting of the wild-typedengue-2 backbone, DEN-2 16681, and the C-prM-E genes of the vaccinestrain of dengue-1, DEN-1 PDK-13.

The preferred chimera of the invention, for example, contains theattenuated dengue-2 virus PDK-53 genome as the viral backbone, in whichthe structural protein genes encoding C, prM and E proteins of thePDK-53 genome, or combinations thereof, are replaced with thecorresponding structural protein genes from a flavivirus to be protectedagainst, such as a different flavivirus or a different dengue virusstrain. Newly discovered flaviviruses or flavivirus pathogens can alsobe incorporated into the DEN-2 backbone. Genetic recombinations withrelated viruses such as hepatitis C virus (HCV) could also be used toproduce the chimeras of this invention. The resulting viral chimera hasthe functional properties of the attenuated dengue-2 virus and istherefore avirulent, but expresses antigenic epitopes of the structuralgene products and is therefore immunogenic.

Nine nucleotide mutations between the genomes of the wild type DEN-216681 virus and two attenuated PDK-53 virus strains are identifiedherein (Table 3). Three of these mutations are silent mutations in thatthey do not result in the production of an amino acid that differs fromthe amino acid in the same position in the wild type virus. The firstmutation is a C-to-T (wild type-to-PDK-53) nucleotide mutation at genomenucleotide position 57 (nt 57) in the 5′ noncoding region. The secondmutation is a A-to-T mutation at genome nucleotide position 524,encoding the amino acid substitution Asp-to-Val in the structuralprotein premembrane region, prM-29.

In the nonstructural protein regions, a Gly-to-Asp (wild type-to-PDK-53)mutation was discovered at nonstructural protein NS1-53 (genomenucleotide position 2579); a Leu-to-Phe (wild type-to-PDK-53) mutationwas discovered at nonstructural protein NS2A-181 (genome nucleotideposition 4018); a Glu-to-Val (wild type-to-PDK-53) mutation wasdiscovered at nonstructural protein NS3-250 (genome nucleotide position5270); and a Gly-to-Ala mutation (wild type-to-PDK-53) was discovered atnonstructural protein NS4A-75 (genome nucleotide position 6599).

The attenuated PDK-53 virus strain has a mixed genotype at genome nt5270. A significant portion (approximately 29%) of the virus populationencodes the non-mutated NS3-250-Glu that is present in the wild typeDEN-2 16681 virus rather than the NS3-250-Val mutation. As both geneticvariants are avirulent, this mutation may not be necessary in anavirulent chimera.

It was unexpectedly discovered that the avirulence of the attenuatedPDK-53 virus strain can be attributed to the presence of specificmutations in the nucleotide sequence encoding nonstructural proteins andin the 5′ noncoding region (Example 5). In particular, a single mutationat NS1-53, a double mutation at NS1-53 and at 5′NC-57, a double mutationat NS1-53 and at NS3-250 and a triple mutation at NS1-53, at 5′NC-57 andat NS3-250, result in attenuation of the DEN-2 virus. Therefore, thegenome of any dengue-2 virus containing such non-conservative amino acidsubstitutions or nucleotide substitutions at these loci can be used asthe backbone in the avirulent chimeras described herein. The backbonecan also contain further mutations to maintain stability of theavirulent phenotype and to reduce the possibility that the avirulentvirus or chimera might revert back to the virulent wild-type virus. Forexample, a second mutation in the stem of the stem/loop structure in the5′ noncoding region will provide additional avirulent phenotypestability, if desired. The stem of the stem-loop structure is composedof nucleotide residues 11-16 (CUACGU) (SEQ ID NO:29) and nucleotideresidues 56-61 (ACGUAG) (SEQ ID NO:30) of the dengue-2 virus RNA sensesequence, wherein the underlined nucleotide is C in wild-type DEN-216681 virus and U in PDK-53 virus. Mutations to this region disruptpotential secondary structures important for viral replication. Inparticular, mutations in the 5′ noncoding region have the ability todisrupt the function of the positive-sense RNA strand and the functionof the negative-sense strand during replication. A single mutation inthis short (only 6 nucleotide residues in length) stem structure in bothDEN and Venezuelan equine encephalitis viruses disrupts the formation ofthe hairpin structure (Kinney et al., Virology 67, 1269-1277, (1993)).Further mutations in this stem structure decrease the possibility ofreversion at this locus, while maintaining virus viability. Furthermore,flavivirus genomes containing an analogous stem structure that consistsof short nucleotide sequences (stems consisting of 6 or more base pairsin the stem-loop structure) located in the 5′ noncoding region andhaving one or more mutations in the stem structure may also be useful asthe backbone structure of this invention.

Such mutations may be achieved by site-directed mutagenesis usingtechniques known to those skilled in the art. Furthermore, otherflavivirus genomes containing analogous mutations at the same loci afteramino acid sequence alignment, can be used as the backbone structure ofthe chimera of this invention and are defined herein as being equivalentto the dengue-2 PDK-53 genome. It will be understood by those skilled inthe art that the virulence screening assays, as described herein and asare well known in the art, can be used to distinguish between virulentand avirulent backbone structures.

Construction of Flavivirus Chimeras

The flavivirus chimeras described herein can be produced by splicing oneor more of the structural protein genes of the flavivirus against whichimmunity is desired into a PDK-53 dengue virus genome backbone, or theequivalent thereof as described above, using recombinant engineeringtechniques well known to those skilled in the art to remove thecorresponding PDK-53 gene and replace it with the desired gene.Alternatively, using the sequences provided in the sequence listing, thenucleic acid molecules encoding the flavivirus proteins may besynthesized using known nucleic acid synthesis techniques and insertedinto an appropriate vector. Avirulent, immunogenic virus is thereforeproduced using recombinant engineering techniques known to those skilledin the art.

As mentioned above, the gene to be inserted into the backbone encodes aflavivirus structural protein. Preferably the flavivirus gene to beinserted is a gene encoding a C protein, a PrM protein and/or an Eprotein. The sequence inserted into the dengue-2 backbone can encodeboth the PrM and E structural proteins. The sequence inserted into thedengue-2 backbone can encode the C, prM and E structural proteins. Thedengue virus backbone is the PDK-53 dengue-2 virus genome and includeseither the spliced genes that encode the C, PrM and/or E structuralproteins of dengue-1 (DEN-2/1), the spliced genes that encode the PrMand/or E structural proteins of dengue-3 (DEN-2/3), or the spliced genesencode the PrM and/or E structural proteins of dengue-4 (DEN-2/4). In aparticular embodiment of this invention, the spliced gene that encodesthe structural protein of dengue-3 virus directs the synthesis of an Eprotein that contains a leucine at amino acid position 345.

In a particular embodiment, the chimera of this invention encodes the Cstructural protein of dengue-2 virus and directs the synthesis of a Cprotein that contains a serine at amino acid position 100 and comprisesa spliced gene encoding the structural proteins of dengue-4 whichdirects the synthesis of an E protein that contains a leucine at aminoacid position 447.

In a further embodiment, the chimera of this invention encodes the Cstructural protein of dengue-2 virus and directs the synthesis of a Cprotein that contains a serine at amino acid position 100 and comprisesa spliced gene encoding the structural proteins of dengue-4 whichdirects the synthesis of an E protein that contains a leucine at aminoacid position 447 and a valine at amino acid position 364. Thestructural proteins described herein can be present as the onlyflavivirus structural protein or in any combination of flavivirusstructural proteins in a viral chimera of this invention.

The chimeras of this invention are engineered by recombination of fullgenome-length cDNA clones derived from both DEN-2 16681 wild type virusand either of the PDK-53 dengue-2 virus variants (-E or -V(SEQ IDNO:15)). The uncloned PDK-53 vaccine contains a mixture of two genotypicvariants, designated herein as PDK53-E and PDK53-V. The PDK53-V variantcontains all nine PDK-53 vaccine-specific nucleotide mutations,including the Glu-to-Val mutation at amino acid position NS3-250. ThePDK53-E variant contains eight of the nine mutations of the PDK-53vaccine and the NS3-25D-Glu of the parental 16681 virus. Infectious cDNAclones are constructed for both variants, and viruses derived from bothclones are attenuated in mice. The phenotypic markers of attenuation ofDEN-2 PDK-53 virus include small plaque size, temperature sensitivity(particularly in LLC-MK₂ cells), limited replication (particularly inC6/36 cells), attenuation for newborn mice (specifically loss ofneurovirulence for suckling mice) and decreased incidence of viremia inmonkeys. The chimeras that are useful as vaccine candidates areconstructed in the genetic backgrounds of the two DEN-2 PDK-53 variantswhich all contain mutations in nonstructural regions of the genome,including 5′NC-57 C-to-T (16681-to-PDK-53) in the 5′ noncoding region,as well as mutations in the amino acid sequence of the nonstructuralproteins, such as, for example, NS1-53 Gly-to-Asp and NS3-250Glu-to-Val.

Suitable chimeric viruses or nucleic acid chimeras containing nucleotidesequences encoding structural proteins of other flaviviruses or denguevirus serotypes can be evaluated for usefulness as vaccines by screeningthem for the foregoing phenotypic markers of attenuation that indicateavirulence and by screening them for immunogenicity. Antigenicity andimmunogenicity can be evaluated using in vitro or in vivo reactivitywith flavivirus antibodies or immunoreactive serum using routinescreening procedures known to those skilled in the art.

Flavivirus Vaccines

The preferred chimeric viruses and nucleic acid chimeras provide live,attenuated viruses useful as immunogens or vaccines. In a preferredembodiment, the chimeras exhibit high immunogenicity while at the sametime producing no dangerous pathogenic or lethal effects.

Until now, an effective vaccine against all strains of dengue virus hasbeen unavailable. Individual live attenuated vaccine candidates for allfour serotypes have been developed by serial passage of wild-typeviruses in primary dog kidney (PDK) cells or other cell types. Asdescribed above, the PDK-53 virus is a useful dengue vaccine candidate.However, a vaccine derived from PDK-53 would only provide immunityagainst the DEN-2 serotype.

To prevent the possible occurrence of DHF/DSS in patients vaccinatedagainst only one serotype of dengue virus, a tetravalent vaccine isneeded to provide simultaneous immunity for all four serotypes of thevirus. A tetravalent vaccine is produced by combining dengue-2 PDK-53with the dengue-2/1, dengue-2/3, and dengue-2/4 chimeras described abovein a suitable pharmaceutical carrier for administration as a multivalentvaccine.

The chimeric viruses or nucleic acid chimeras of this invention cancomprise the structural genes of either wild-type or attenuated virus ina virulent or an attenuated DEN-2 virus backbone. For example, thechimera may express the structural protein genes of wild-type DEN-116007 virus or its candidate PDK-13 vaccine derivative in either of theDEN-2 PDK-53 backgrounds.

As described in Example 1, all of the chimeric DEN-2/1 virusescontaining the C, prM and E proteins of either DEN-1 16007 virus(DEN-2/1-EP and -VP chimeras) or PDK-13 virus (DEN-2/1-EV and -VV (SEQID NO:7) chimeras) in the backbones of DEN-2 PDK-53 retain all of thephenotypic attenuation markers of the DEN-2 PDK-53 virus. The chimericDEN-2/1-EP and -VP(SEQ ID NO:5) viruses, which contain the C, prM and Eproteins of DEN-1 16007 virus are more genetically stable after passingin cell culture than the DEN-2/1-EV and -VV viruses. The immunogenicityof the chimeric viruses expressing the structural proteins of DEN-116007 virus was higher as compared with the neutralizing antibody titerselicited by the PDK-13 vaccine virus and the chimeras expressing thestructural proteins of the PDK-13 virus. Thus, the chimeric DEN-2/1-EPand -VP viruses, which express the structural genes of wild-type DEN-116007 virus within the genetic background of the two DEN-2 PDK-53variants, are potential DEN-1 vaccine candidates that are superior tothe candidate PDK-13 vaccine. These two chimeras replicate well inLLC-MK₂ cells and retain the attenuation markers associated with DEN-2PDK-53 virus, including small plaque size, temperature sensitivity,restricted replication in mosquito cells and attenuation for mice. Theyare at least as immunogenic as wild-type DEN-1 16007 virus in mice.

Other examples, such as DEN-2/3 and DEN-2/4 chimeras, in Examples 2-4,also showed that chimeric viruses containing structural protein genesfrom wild-type DEN-3 or DEN-4 virus within the DEN-2 PDK-53 backbones,are suitable vaccine candidates which retain all of the attenuatedphenotypic markers of the DEN-2 PDK-53 viruses (Table 14), whileproviding immunogenicity against DEN-3 or DEN-4 virus. The strategydescribed herein of using a genetic background that contains thedeterminants of attenuation in nonstructural regions of the genome toexpress the structural protein genes of heterologous viruses has lead todevelopment of live, attenuated flavivirus vaccine candidates thatexpress wild-type structural protein genes of optimal immunogenicity.Thus, vaccine candidates for immunogenic variants of multiple flaviviralpathogens can be designed.

Viruses used in the chimeras described herein are typically grown usingtechniques known in the art. Virus plaque titrations are then performedand plaques counted in order to assess the viability and phenotypiccharacteristics of the growing cultures. Wild type viruses are passagedthrough cultured cell lines to derive attenuated candidate startingmaterials.

Chimeric infectious clones are constructed from the various dengueserotype clones available. The cloning of virus-specific cDNA fragmentscan also be accomplished, if desired. The cDNA fragments containing thestructural protein or nonstructural protein genes are amplified byreverse transcriptase-polymerase chain reaction (RT-PCR) from denguevirus RNA with various primers. Amplified fragments are cloned into thecleavage sites of other intermediate clones. Intermediate, chimericdengue virus clones are then sequenced to verify the accuracy of theinserted dengue virus-specific cDNA.

Full genome-length chimeric plasmids constructed by inserting thestructural protein or nonstructural protein gene region of dengueserotype viruses into vectors are obtainable using recombinanttechniques well known to those skilled in the art.

Nucleotide and Amino Acid Analysis

The nucleotide sequence for DEN-2 16681 and corresponding PDK-53-V areprovided in SEQ ID NO:13 and SEQ ID NO:15, respectively. Amino acidsequences for DEN-2 16681 and corresponding PDK-53-V are provided in SEQID NO:14 and SEQ ID NO:16. The -E variant of PDK-53, which varies fromPDK-53-V at nucleotide position 5270 and amino acid position 1725 of thepolyprotein is further described in Table 3. A comparison of thecritical nucleotide and amino acid substitutions that have beendiscovered between the parent strain and the attenuated virus is shownin Table 3. The sequence of the DEN-2 cDNA amplicons was amplified fromDEN-2 viral genomic RNA by reverse transcriptase-polymerase chainreaction (RT-PCR).

Unlike PDK-53, which contains no amino acid mutations in the E proteinrelative to wild type dengue-2 virus, the Mahidol DEN-1, DEN-3 and DEN-4attenuated viruses all have amino acid mutations in the E protein(Tables 2, 4 & 5). The wild-type DEN-3 16562 listed in the sequencelisting (nucleotide sequence, SEQ ID NO:19; amino acid sequence, SEQ IDNO:20) was shown to comprise traces of a variant comprising a T atnucleotide position 1521 which directs incorporation of a leucine atpolyprotein position 476, amino acid residue position 476 of the Eprotein.

Each of the latter three viruses possess a Glu-to-Lys(parent-to-vaccine) mutation in the E protein, although the mutation islocated at a different amino acid residue in the E protein. Thissubstitution causes a shift from a negatively charged amino acid to apositively charged one. The Glu-to-Lys substitution in the E protein ofDEN-4 vaccine virus was the only mutation present in the E protein,while the E proteins of DEN-1 and DEN-3 vaccine viruses had five andthree amino acid mutations, respectively.

The NS1-53 mutation in the DEN-2 PDK-53 vaccine virus is significant forthe attenuated phenotype of this virus, because the NS1-53-Gly of theDEN-2 16681 virus is conserved in nearly all flaviviruses, including thetick-borne viruses, sequenced to date. The Mahidol DEN-4 vaccine virusalso contains an amino acid mutation in the NS1 protein at position 253.This locus, which is a Gln-to-His mutation in DEN-4 PDK-48 vaccine virusis Gln in all four wild serotypes of dengue virus. This Gln residue isunique to the dengue viruses within the flavivirus genus. The NS1protein is a glycoprotein that is secreted from flavivirus-infectedcells. It is present on the surface of the infected cell andNS1-specific antibodies are present in the serum of virus-infectedindividuals. Protection of animals immunized with NS1 protein orpassively with NS1-specific antibody has been reported. The NS1 proteinappears to participate in early viral RNA replication.

The mutations that occurred in the NS2A, NS2B, NS4A, and NS4B proteinsof the DEN-1, -2, -3 and -4 attenuated strains were all conservative innature. The NS4A-75 and NS4A-95 mutations of DEN-2 and DEN-4 vaccineviruses, respectively, occurred at sites of amino acid conservationamong dengue viruses, but not among flaviviruses in general.

The flaviviral NS3 protein possesses at least two recognized functions:the viral proteinase and RNA helicase/NTPase. The 698-aa long (DEN-2virus) NS3 protein contains an amino-terminal serine protease domain(NS3-51-His, -75-Asp, -135-Ser catalytic triad) that is followed bysequence motifs for RNA helicase/NTPase functions (NS3-196-GAGKT (SEQ IDNO:147), -284-MAIL -459-GRIGR (SEQ ID NO:148)). None of the mutations inthe NS3 proteins of DEN-1, DEN-2, or DEN-3 virus occurred within arecognized motif. The NS3-510 Tyr-to-Phe mutation in DEN-1 PDK-13 viruswas conservative. Since the wild-type DEN-2, -3 and -4 viruses containPhe at this position, it is unlikely that the Tyr-to-Phe mutation playsa role in the attenuation of DEN-1 virus. The NS3-182 Glu-to-Lysmutation in DEN-1 PDK-13 virus occurred at a position that is conservedas Asp or Glu in most mosquito-borne flaviviruses and it may play somerole in attenuation. This mutation was located 15 amino acid residuesupstream of the GAGKT (SEQ ID NO:147) helicase motif. As noted inprevious reports, the NS3-250-Glu in DEN-2 16681 virus is conserved inall mosquito-borne flaviviruses except for yellow fever virus.

Method of Administration

The viral chimeras described herein are individually or jointly combinedwith a pharmaceutically acceptable carrier or vehicle for administrationas an immunogen or vaccine to humans or animals. The terms“pharmaceutically acceptable carrier” or “pharmaceutically acceptablevehicle” are used herein to mean any composition or compound including,but not limited to, water or saline, a gel, salve, solvent, diluent,fluid ointment base, liposome, micelle, giant micelle, and the like,which is suitable for use in contact with living animal or human tissuewithout causing adverse physiological responses, and which does notinteract with the other components of the composition in a deleteriousmanner.

The immunogenic or vaccine formulations may be conveniently presented inviral PFU unit dosage forth and prepared by using conventionalpharmaceutical techniques. Such techniques include the step of bringinginto association the active ingredient and the pharmaceutical carrier(s)or excipient(s). In general, the formulations are prepared by uniformlyand intimately bringing into association the active ingredient withliquid carriers. Formulations suitable for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe formulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example, sealed ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for example,water for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets commonly used by one of ordinary skill in the art.

Preferred unit dosage formulations are those containing a dose or unit,or an appropriate fraction thereof, of the administered ingredient. Itshould be understood that in addition to the ingredients particularlymentioned above, the formulations of the present invention may includeother agents commonly used by one of ordinary skill in the art.

The immunogenic or vaccine composition may be administered throughdifferent routes, such as oral or parenteral, including, but not limitedto, buccal and sublingual, rectal, parenteral, aerosol, nasal,intramuscular, subcutaneous, intradermal, and topical. The compositionmay be administered in different forms, including, but not limited to,solutions, emulsions and suspensions, microspheres, particles,microparticles, nanoparticles and liposomes. It is expected that fromabout 1 to about 5 doses may be required per immunization regimen.Initial doses may range from about 100 to about 50,000 PFU, with apreferred dosage range of about 500 to about 20,000 PFU, a morepreferred dosage range of from about 1000 to about 12,000 PFU and a mostpreferred dosage range of about 1000 to about 4000 PFU. Boosterinjections may range in dosage from about 100 to about 20,000 PFU, witha preferred dosage range of about 500 to about 15,000, a more preferreddosage range of about 500 to about 10,000 PFU, and a most preferreddosage range of about 1000 to about 5000 PFU. For example, the volume ofadministration will vary depending on the route of administration.Intramuscular injections may range in volume from about 0.1 ml to 1.0

The composition may be stored at temperatures of from about −100° C. toabout 4° C. The composition may also be stored in a lyophilized state atdifferent temperatures including room temperature. The composition maybe sterilized through conventional means known to one of ordinary skillin the art. Such means include, but are not limited to, filtration. Thecomposition may also be combined with bacteriostatic agents, such asthimerosal, to inhibit bacterial growth.

Administration Schedule

The immunogenic or vaccine composition described herein may beadministered to humans, especially individuals traveling to regionswhere dengue virus infection is present, and also to inhabitants ofthose regions. The optimal time for administration of the composition isabout one to three months before the initial infection. However, thecomposition may also be administered after initial infection toameliorate disease progression, or after initial infection to treat thedisease.

Adjuvants

A variety of adjuvants known to one of ordinary skill in the art may beadministered in conjunction with the chimeric virus in the immunogen orvaccine composition of this invention. Such adjuvants include, but arenot limited to, the following: polymers, co-polymers such aspolyoxyethylene-polyoxypropylene copolymers, including blockco-polymers, polymer P1005, Freund's complete adjuvant (for animals),Freund's incomplete adjuvant; sorbitan monooleate, squalene, CRL-8300adjuvant, alum, QS 21, muramyl dipeptide, CpG oligonucleotide motifs andcombinations of CpG oligonucleotide motifs, trehalose, bacterialextracts, including mycobacterial extracts, detoxified endotoxins,membrane lipids, or combinations thereof.

Nucleic Acid Sequences

Nucleic acid sequences of the DEN-1 16007 (SEQ ID NO:1), DEN-1 PDK-13(SEQ ID NO:3), DEN-2 16681 (SEQ ID NO:13), DEN-2 PDK-53 (SEQ ID NO:15),DEN-3 16562 (SEQ ID NO:19), DEN-3 PGMK-30/FRhL-3 (SEQ ID NO:21), DEN-41036 (SEQ ID NO:23) and DEN-4 PDK-13 (SEQ ID NO:25) viruses are usefulfor designing nucleic acid probes and primers for the detection ofdengue virus in a sample or specimen with high sensitivity andspecificity. Probes or primers corresponding to each viral subtype canbe used to detect the presence of DEN-1 virus, DEN-3 virus and DEN-4virus, respectively, to detect dengue virus infection in general in thesample, to diagnose infection with dengue virus, to distinguish betweenthe various dengue virus subtypes, to quantify the amount of denguevirus in the sample, or to monitor the progress of therapies used totreat a dengue virus infection. The nucleic acid and corresponding aminoacid sequences are also useful as laboratory research tools to study theorganisms and the diseases and to develop therapies and treatments forthe diseases.

Nucleic acid probes selectively hybridize with nucleic acid moleculesencoding the DEN-1, DEN-3 and DEN-4 viruses or complementary sequencesthereof. By “selective” or “selectively” is meant a sequence which doesnot hybridize with other nucleic acids to prevent adequate detection ofthe dengue virus. Therefore, in the design of hybridizing nucleic acids,selectivity will depend upon the other components present in a sample.The hybridizing nucleic acid should have at least 70% complementaritywith the segment of the nucleic acid to which it hybridizes. As usedherein to describe nucleic acids, the term “selectively hybridizes”excludes the occasional randomly hybridizing nucleic acids, and thus,has the same meaning as “specifically hybridizing.” The selectivelyhybridizing nucleic acid of this invention can have at least 70%, 80%,85%, 90%, 95%, 97%, 98%, and 99% complementarity with the segment of thesequence to which it hybridizes, preferably 85% or more.

The present invention also contemplates sequences, probes and primerswhich selectively hybridize to the encoding nucleic acid or thecomplementary, or opposite, strand of the nucleic acid. Specifichybridization with nucleic acid can occur with minor modifications orsubstitutions in the nucleic acid, so long as functionalspecies-specific hybridization capability is maintained. By “probe” ismeant nucleic acid sequences that can be used as probes or primers forselective hybridization with complementary nucleic acid sequences fortheir detection or amplification, which probes can vary in length fromabout 5 to 100 nucleotides, or preferably from about 10 to 50nucleotides, or most preferably about 18-24 nucleotides. Therefore, theterms “probe” or “probes” as used herein are defined to include“primers.” Isolated nucleic acids are provided herein that selectivelyhybridize with the species-specific nucleic acids under stringentconditions and should have at least five nucleotides complementary tothe sequence of interest as described in Molecular Cloning: A LaboratoryManual, 2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., (1989).

If used as primers, the composition preferably includes at least twonucleic acid molecules which hybridize to different regions of thetarget molecule so as to amplify a desired region. Depending on thelength of the probe or primer, the target region can range between 70%complementary bases and full complementarity and still hybridize understringent conditions. For example, for the purpose of detecting thepresence of the dengue virus, the degree of complementarity between thehybridizing nucleic acid (probe or primer) and the sequence to which ithybridizes is at least enough to distinguish hybridization with anucleic acid from other organisms.

The nucleic acid sequences encoding the DEN-1, DEN-3 or DEN-4 virus canbe inserted into a vector, such as a plasmid, and recombinantlyexpressed in a living organism to produce recombinant dengue viruspeptides and/or polypeptides.

Nucleic Acid Detection Methods

A rapid genetic test that is diagnostic for each of the vaccine virusesdescribed herein is provided by the current invention. This embodimentof the invention enhances analyses of viruses isolated from the serum ofvaccinated humans who developed a viremia, as well as enhancingcharacterization of viremia in nonhuman primates immunized with theMahidol candidate vaccine viruses.

As provided in the complete nucleotide sequences of the wild-typeparental and vaccine strains, and in the primer sequences provided inTable 16, the current invention comprises viruses specific probes andprimers to detect one or more of the mutations that have been identifiedin the genome of each vaccine virus. Specific detection of two or morevirus specific loci allows specific identification of the particularvaccine circulating in the serum of a vaccinee. Examples of such probesspecifically designed to allow detection of each of the DEN-1, DEN-2,DEN-3 and DEN-4 vaccine virus-specific loci in a TaqMan assay areprovided in Table 16.

These sequences include a diagnostic TaqMan probe that serves to reportthe detection of the cDNA amplicon amplified from the viral genomic RNAtemplate by using a reverse-transcriptase/polymerase chain reaction(RT/PCR), as well as the forward (F in Table 16) and reverse (R in Table16) amplimers that are designed to amplify the cDNA amplicon, asdescribed below. In certain instances, one of the amplimers has beendesigned to contain a vaccine virus-specific mutation (underlinedresidues in Table 16) at the 3′-terminal end of the amplimer, whicheffectively makes the test even more specific for the vaccine strainbecause extension of the primer at the target site, and consequentlyamplification, will occur only if the viral RNA template contains thatspecific mutation. The probes and primers listed in Table 15 and Table16 serve as examples of useful diagnostic sequences and are not intendedto limit the design or scope of other probe and amplimer sequences thatmight be designed to detect the Mahidol vaccine virus-specific geneticmutations.

A recently developed, automated PCR-based nucleic acid sequencedetection system is the TaqMan assay (Applied Biosystems), which isbecoming widely used in diagnostic laboratories. The TaqMan assay is ahighly specific and sensitive assay that permits automated, real timevisualization and quantitation of PCR-generated amplicons from a samplenucleic acid template. TaqMan can determine the presence or absence of aspecific sequence. In this assay, a forward and a reverse primer aredesigned to anneal upstream and downstream of the target mutation site,respectively. A specific detector probe, which is designed to have amelting temperature of about 10° C. higher than either of the amplimersand containing the vaccine virus-specific nucleotide mutation or itscomplement (depending on the strand of RT/PCR amplicon that is beingdetected), constitutes the third primer component of this assay.

The probe is a fluorescent detector or reporter oligonucleotide thatcontains a 5′-reporter dye and a 3′-quencher dye. The 5′ end of thenucleotide is linked to one of a number of different fluorescentreporter dyes, such as FAM (6-carboxyfluorescein) or TET(tetrachloro-6-carboxyfluorescein). At the 3′-end of the probe, thequenching dye TAMRA (6-carboxytetramethylrhodamine) is attached via alinker. The quenching dye suppresses the fluorescence of the reporterdye in the intact probe, where both dyes are in close proximity. If theprobe-specific target sequence is present in the RT/PCR amplicon, theprobe will anneal between the forward and reverse primer sites in theamplicon. If the probe hybridizes to the target sequence, the 5′-3′nuclease activity of AmpliTaq Gold DNA polymerase (Applied Biosystems)cleaves the probe between the reporter dye and the quencher dye. Thepolymerase will not digest free probe. Because the polymerase displacesthe probe, polymerization and PCR amplification continue. Once separatedfrom the quencher dye, the reporter dye produces a fluorescence that ismeasured by the ABI PRISM Sequence Detection System. If theprobe-specific target sequence is present in the amplicon, the level offluorescence increases with, and is automatically measured at, eachamplifying PCR cycle.

A probe designed to specifically detect a mutated locus in one of theMahidol vaccine viral genomes will contain the vaccine-specificnucleotide in the middle of the probe. This probe will result indetectable fluorescence in the TaqMan assay if the viral RNA template isvaccine virus-specific. However, genomic RNA templates from wild-typeDEN viruses will have decreased efficiency of probe hybridizationbecause of the single nucleotide mismatch (in the case of the parentalviruses DEN viruses) or possibly more than one mismatch (as may occur inother wild-type DEN viruses) and will not result in significantfluorescence. The DNA polymerase is more likely to displace a mismatchedprobe from the RT/PCR amplicon template than to cleave the mismatchedprobe to release the reporter dye (TaqMan Allelic Discrimination assay,Applied Biosystems).

A more recently developed strategy for diagnostic genetic testing makesuse of molecular beacons (Tyagi and Kramer, Nature Biotechnology14:303-308 (1996)). The molecular beacon strategy also utilizes primersfor RT/PCR amplification of amplicons, and detection of a specificsequence within the amplicon by a probe containing reporter and quencherdyes at the probe termini. In this assay, the probe forms a stem-loopstructure. The 5′- and 3′-terminal reporter dye and quencher dye,respectively, are located at the termini of the short stem structure,which brings the quencher dye in close juxtaposition with the reporterdye. The stem-structure is melted during the denaturation step of theRT/PCR assay. If the target viral RNA contains the target sequence andis amplified by the forward and reverse amplimers, the opened loop ofthe probed hybridizes to the target sequence during the annealing stepof the cycle. When the probe is annealed to either strand of theamplicon template, the quencher and reporter dyes are separated, and thefluorescence of the reporter dye is detected. This is a real-timeidentification and quantitation assay that is very similar to the TaqManassay. The molecular beacons assay employs quencher and reporter dyesthat differ from those used in the TaqMan assay.

The present invention is further illustrated by the followingnon-limiting examples, which are not to be construed in any way asimposing limitations upon the scope thereof. On the contrary, it is tobe clearly understood that resort may be had to various otherembodiments, modifications, and equivalents thereof which, after readingthe description herein, may suggest themselves to those skilled in theart without departing from the spirit of the present invention or thescope of the appended claims.

Example 1 Preparation of a Chimeric Dengue-1 Vaccine PDS-53/Dengue-1Viruses and Cell Cultures

Wild-type DEN-1 16007 and DEN-2 16681 viruses were available in thevirus collection at the Centers for Disease Control and Prevention(Atlanta, Ga.). DEN-1 16007 virus was recovered from the serum of apatient with DHF/DSS in 1964 in Thailand. The virus was isolatedfollowing three passages in grivet monkey kidney BS-C-1 cells and onepassage in LLC-MK₂ cells, passaged twice in Toxorhynchites amboinensismosquitoes, and then passaged in primary dog kidney (PDK) cells at theCenter for Vaccine Development, Mahidol University, Thailand, to derivethe candidate DEN-1 PDK-13 vaccine virus (Yoksan et al., “Dengue virusvaccine development: study on biological markers of uncloned dengue 1-4viruses serially passaged in primary kidney cells,” pp. 35-38, InArbovirus research in Australia, Proceedings of the Fourth Symposium.CSIRO/QIMR, Brisbane, Australia (1986); Bhamarapravati & Sutee, Vaccine18: 44-47 (2000)). A single LLC-MK₂ passage of this candidate vaccinevirus (lot Mar. 10, 1989) was used, unless otherwise mentioned.Following the aforementioned mosquito passages, the 16007 virus waspassaged once in LLC-MK₂ cells for use.

Viruses were grown in LLC-MK₂ and C6/36 cells in Dulbecco's modifiedminimal essential medium (DMEM) containing penicillin/streptomycin and5% fetal bovine serum (FBS). Virus plaque titrations were performed in6-well plates of confluent Vero or LLC-MK₂ cells as described previously(Miller et al., Am. Trop. Med. & Hyg. 35: 1302-1309 (1986)). The first4-ml overlay medium-containing 1% SeaKem LE agarose (FMC BioProducts,Rockland, Me.) in nutrient medium (0.165% lactalbumin hydrolysate [DifcoLaboratories, Detroit, Mich.]), 0.033% yeast extract [Difco], Earl'sbalanced salt solution, 25 mg/L of gentamycin sulfate [Bio Whittaker,Walkersville, Md.], 1.0 mg/L of amphotericin B [Fungizone, E. R. Squibb&Sons, Princeton, N.J.], and 2% FBS)—was added after adsorption of the200-μl virus inoculum for 1.5 h at 37° C. Following incubation at 37° C.for 7 days, a second 2-ml overlay containing an additional 80 μg/ml ofneutral red vital stain (GIBCO-BRL, Gaithersburg, Md.) was added.Plaques were counted 8 to 11 days after infection.

Construction of Chimeric D2/1 Infectious Clones

pD2-16681-P48, pD2-PDK53-E48, pD2-PDK53-V48 Vectors

Three DEN-2 vectors were used for construction of the chimeric D2/1clones. These were modified from the DEN-2 infectious clones reported byKinney et al. (Virology 230: 300-308 (1997)). Clone pD2-16681-P48 wasmodified from pD2/IC-30P-A to contain cloning sites MluI and NgoMIV atnucleotide positions 451 and 2380, respectively. The same cloning siteswere introduced into both DEN-2 PDK-53 virus-specific clones,pD2/IC-130Vx-4 and -130Vc-K, and the modified clones were designated aspD2-PDK53-E48 and pD2-PDK53-V48, respectively. Two cloning errors werefound in the original pD2/16-130Vx-4 and -130Vc-IC at nt-6665 andnt-8840. These defects were corrected in pD2-PDK53-E48 and -V48. Theintroduced NgoMIV cloning site resulted in two nucleotide mutations (nt2381 and 2382; TG to CC), which encoded a Val-to-Ala substitution atE-482. The nucleotide changes introduced at the MluI site were silent.The Maur site introduced at the C/prM junction was used to clone theprM-E genes of heterologous viruses.

Chimeric pD2/1-PP, -EP, -VP, -PV, -EV, and -VV

Two intermediate DEN-2 clones, pD2I-P and pD2I-E, were constructed bydeleting the HpaI (nt-2676) to XbaI (3′ terminus of viral genomic cDNA)fragments of pD2-16681-P48 and pD2-PDK53-E48, respectively. Theseintermediate clones were used to subclone DEN-1 virus-specific cDNAfragments. The cDNA fragments containing the C-prM-E genes of DEN-116007or PDK-13 virus were amplified by reverse transcriptase-polymerase chainreaction (RT-PCR) from DEN-1 viral RNA with primers DEN-Bg1.5NC(5′-TAGAGAGCAGATCTCTG-3′ (SEQ ID NO:31); conserved sequence in the 5′NCRof dengue viral genomes, underlined sequence is a BglII site) andcD1-2394.Ngo:

(SEQ ID NO: 32) (5′-TGTGACCATGCCGGCTGCGATGCACTCACCGA-3′; underlined NgoMIV site followed by complementary sequence near the 3′end of the E gene of DEN-1 virus). Amplified fragments were cloned intothe BglII-NgoMIV sites of the intermediate pD2I-P and pD2I-E clones.Intermediate, chimeric pD2/1 clones were sequenced to verify theaccuracy of the inserted DEN-1 virus-specific cDNA. Fragments excisedfrom the intermediate pD2/1 clones with SstI (preceding the T7 promoter)and NgoMIV were cloned into the full genome-length DEN-2 vectors,pD2-16681-P48, pD2-PDK53-E48, and pD2-PDK53-V48. Six full genome-lengthchimeric pD2/1 plasmids were constructed by inserting the C-prM-E generegion of DEN-1 16007 or PDK-13 virus into these three vectors (FIG. 1).The plasmids were designated pD2/1-XY and their virus derivatives weredesignated DEN2/1-XY, where X=the infectious DEN-2 clone background(P=parental 16881 clone, E=PDK53-E variant, V=PDK53-V variant) andY=DEN-1 virus-specific C-prM-E insert (P=parental 16007 strain, V=PDK-13vaccine candidate). The PDK53-E backbone contains three DEN-2 PDK-53virus-specific amino acid mutations (NS1-53-Asp, NS2A-181-Phe andNS4A-75-Ala) as well as the 5′NC-57 mutation of PDK-53 virus. ThePDK53-V backbone contains these same PDK-53 virus-specific loci plus theadditional PDK-53 virus-specific NS3-250-Val locus.

Recovery of Recombinant Viruses

All recombinant plasmids were grown in Escherichia coli XL1-Blue cells.Recombinant viral RNA was transcribed and capped with the cap analog m⁷GpppA from 200-400 ng of XbaI-linearized cDNA, and transfected into3-4×10⁶ LLC-MK₂ or BHK-21 cells by electroporation. Transfected cellswere transferred to 75-cm² flasks in DMEM medium containing 10% FBS.Viral proteins expressed in the transfected cells were analyzed byindirect immunofluorescence assay (IFA). Virus-infected cells were fixedin ice-cold acetone for 30 min. DEN-1 and DEN-2 virus-specificmonoclonal antibodies 1F1 and 3H5, respectively, were used in the assay,and binding was detected with fluorescein-labeled goat anti-mouseantibody. Viruses were harvested after 8 to 10 days, and were thenpassaged in LLC-MK₂ cells once (DEN-2-16681-P48, DEN-2 PDK53-E48 and-V48, DEN-2/1-PP, -EP, and -VP) or twice (DEN-2/1-PV, -EV, and -VV) toobtain working seeds. D2/1-EV and -VV viruses were passaged a third timein LLC-MK₂ cells to obtain higher viral titers required for challenge orimmunization of mice.

Characterization of the Replication Phenotypes of Chimeric Viruses inCell Cultures

Plaque sizes were measured 10 days after infection in LLC-MK₂ cells.Mean plaque diameters were calculated from 10 plaques for each virus.Viral growth curves were performed in 75-cm² flasks of LLC-MK₂ or C6/36cells at approximately 0.001 multiplicity of infection (m.o.i.). Afteradsorption for 2 h, 30 ml of DMEM medium (for LLC-MK₂ cells) or overlaynutrient medium (for C6/36 cells) containing 5% FBS andpenicillin/streptomycin was added, and the cultures were incubated in 5%CO₂ at 37° C. or 29° C., respectively. Aliquots of culture medium wereharvested at 48-h intervals for 12 days, adjusted to 12.5% FBS, andstored at −80° C. prior to titration.

Temperature sensitivity was tested in LLC-MK₂ cells. Cells grown in twosets of 75-cm² flasks were infected and incubated as described for thegrowth curve study. One set of cultures was incubated for 8 days at 37°C., the other at 38.7° C. The ratio of virus titer at 38.7° C. versusthe titer at 37° C. was calculated. Virus was designated astemperature-sensitive if the virus titer at 38.7° C. was reduced 60% orgreater, relative its titer at 37° C.

Sequencing of Viral cDNA

Viral RNA was extracted from virus seed or by using the QIAmp Viral RNAkit (Qiagen, Valencia, Calif.). DEN-1 virus-specific primers were basedon the published data of the Singapore strain S275/90 (Fu et al.,Virology 88: 953-958 (1992)). Five to 7 overlapping viral cDNA fragmentswere amplified by RT-PCR with the Titan One-Tube RT-PCR System (RocheMolecular Biochemicals, Indianapolis, hid.). Both strands of the cDNAamplicons were sequenced directly. For sequencing of the DEN-1 PDK-13viral genome, template genomic RNA was extracted directly from a vial ofthe candidate DEN-1 PDK-13 vaccine (lot Mar. 10, 1989). The 5′- and3′-terminal sequences of the DEN-1 16007 and DEN-1 PDK-13 viral genomeswere determined with the 5′ RACE kit (GIBCO BRL) and by tailing thegenomic RNA with poly(A). Automated sequencing was performed asrecommended on a PRISM 377 sequencer (Perkin-Elmer/Applied Biosystems,Foster City, Calif.).

Mouse Studies

Litters of newborn, 1-day-old outbred white ICR mice were inoculatedintracranially with 5,000 PFU of virus in a volume of 30 ml. They wereobserved daily for paralysis and death, and surviving mice wereindividually weighed once each week for 5 weeks.

Neutralizing antibody responses were tested in 3-week-old ICR mice. Theywere inoculated intraperitoneally with 10⁴ PFU of virus, and wereboosted with the same amount of virus 3 weeks or 6 weeks later. Micewere bled 2 days prior to the boost and 3 weeks after boosting.

The ICR strain of inbred mice used above are not usually fatallysusceptible to challenge with wild-type DEN-1 virus. Therefore, to fullytest the ability of the DEN-2/1 viral chimera to induce an effectiveimmune response, it was necessary to utilize inbred AG-129 mice, whichlack receptors for both interferon alpha/beta and interferon gamma(Muller et al., Science 264:1918-1921 (1994)), as these mice have beenfound to be susceptible to intraperitoneal challenge with high doses ofwild-type DEN-1 virus, strain Mochizuki. Therefore, 3.5-4.5-week-oldinbred AG-129 mice were immunized intraperitoneally with 10⁴ PFU ofwild-type DEN-1 16007, Mahidol candidate vaccine DEN-1 PDK-13, chimericDEN-2/1-EP or chimeric DEN-2/1-VP virus. These immunized mice werechallenged intraperitoneally with a lethal dose of DEN-1 Mochizukivirus.

Neutralization Assays

Mouse serum samples were tested for neutralizing antibodies byserum-dilution plaque-reduction neutralization test (PRN′T). Sixty PFUof DEN-1 16007 virus was incubated with serial 2-fold dilutions ofheat-inactivated (56° C. for 30 min) mouse serum specimens overnight at4° C. The neutralizing antibody titer was identified as the highestserum dilution that reduced the number of virus plaques in the test by50% or greater.

Results

To assess the potential of infectious cDNA clones derived from the twovariants of DEN-2 16681 PDK-53 virus (PDK-53-E and PDK-53-V) to serve asvectors for vaccine development, we engineered chimeric DEN-2/DEN-1 cDNAclones (D2/1-EP, D2/1-VP, D2/1-EV, and D2/1-VV) containing thestructural genes (C-prM-E) of wild-type DEN-1 16007 virus or its vaccinederivative, strain PDR-13, within the backbone of these two vectors(FIG. 1). Two other chimeric clones, D2/1-PP and D2/1-PV, containing thestructural genes (C-prM-E) of DEN-1 16007 or PDK-13 virus in thebackbone of wild-type DEN-2 16681 virus, were also constructed forcomparison (FIG. 1). We sequenced the entire full-length genomic cDNA inall of the infectious clones. A silent cDNA artifact was incorporatedinto the chimeric clones at nt-297 (T-to-C). A silent mutation atnt-1575 (T-to-C) was engineered into all of the chimeric clones toremove the natural XbaI site in the E gene of the DEN-1 virus.

Titers after transfection of LLC-MK₂ or BHK-21 cells were 10⁶-10⁶ PFU/mlfor the chimeric viruses D2/1-PP, -EP, and -VP containing the C-prM-E ofDEN-1 16007 virus. These titers increased to 10⁶⁵-10^(7.5) PFU/ml aftera single passage in LLC-MK₂ cells, comparable to the titers obtained fortheir parental viruses. Lower titers of 10²-10⁴ PFU/ml were obtained intransfected cells for the chimeric DEN-2/1-PV, -EV, and -VV virusescontaining the C-prM-E of DEN-1 PDK-13 virus. D2/1-PV virus reached 10⁶PFU/ml after 2 passages in LLC-MK₂ cells, whereas D2/1-EV and -VVviruses reached titers of 10″-10⁵³ PFU/ml after two or three passages.Cells infected with any of the chimeric DEN-2/1 viruses were positive byIFA with monoclonal antibody 1F1 (specific for DEN-1 E protein) andnegative with monoclonal antibody 3H5 (specific for DEN-2 E protein),indicating that appropriate DEN-1 E proteins were expressed by thechimeras. The DEN-2/1-PP, DEN-2/1-EP, and DEN-2/1-VP viral genomes werefully sequenced by directly analyzing overlapping RT-PCR fragmentsamplified from genomic viral RNA extracted from master seeds. All threegenomes had the expected sequence.

Growth of the Chimeric Viruses in LLC-MK₂ and C6/36 Cell Cultures

All of the chimeric DEN-2/1 viruses produced smaller plaques, relativeto the 6.8±0 4-mm plaque of wild-type DEN-1 16007 virus in LLC-MK₂ cells(FIG. 2A). Both DEN-2/1-EP (3.1±0.3 mm) and -VP (2.8±0.3 mm) viralplaques were similar in size to those of DEN-1 PDK-13 virus (2.9±10.3mm) The chimeric viruses DEN-2/1-PV, DEN-2/1-EV, and DEN-2/1-VVcontaining the C-prM-E of DEN-1 PDK-13 virus formed tiny (1.3±0.3 mm) orpinpoint (<1 mm) plaques. The DEN-2 16681-P48 virus produced 3.5±0 3-mmplaques that were similar to plaques of wild-type DEN-2 16681 virus. TheDEN-2 PDK53-V48 virus formed plaques that were smaller and fuzzier thanthose of the DEN-2 PDK53-E48 virus. The 5.1±0.3-mm plaques of DEN-2/1-PPvirus were larger than those of the other chimeric viruses, but smallerthan those of DEN-1 16007 virus.

Viruses were tested for temperature sensitivity in LLC-MK₂ cells.Temperature sensitivity was determined on day 8 or 10 after infection asindicated in FIG. 2B.

Temperature sensitivity was based on the reduction of virus titers at38.7° C. from those at 37° C. Temperature sensitivity was calculatedfrom measurements taken in at least 3 experiments.

The DEN-2 PDK53-V variant (D2-PDK53-V48) was more temperature sensitivethan DEN-2 PDK53-E virus (D2-PDK53-E48) and DEN-2 16681 virus(DEN-2-16681-P48) was somewhat temperature sensitive (70-87% titerreduction at 38.7° C.). Multiple temperature sensitivity tests forDEN-2-PDK53-E48 virus resulted in 83%-97% growth reduction. The titer ofDEN-1 16007 virus was reduced by 40% or less at 38.7° C., making it theleast-temperature sensitive virus in this study. All of the chimericviruses were temperature sensitive relative to DEN-1 16007 virus, andwere at least as temperature-sensitive as PDK-13.

All of the DEN-1, DEN-2, and chimeric D2/1 viruses reached peak titersbetween 8 and 10 days after infection in LLC-MK₂ cells (FIG. 2B). Theclone-derived viruses DEN-2-16681-P48, DEN-2-PDK53-E48, andDEN-2-PDK53-V48 replicated to 10″ PFU/ml or greater, as did DEN-2 16681and PDK-53 viruses. Although reaching similar peak titer,DEN-2-PDK53-V48 virus replicated slower than the DEN-2-PDK53-E48 virusduring the first 4 days after infection. Chimeric DEN-2/1-PP,DEN-2/1-EP, DEN-2/1-VP, and DEN-2/1-PV viruses reached peak titers over10^(6.7) PFU/ml, comparable to the peak titers of their parental DEN-1and DEN-2 viruses. Chimeric DEN-2/1-EV and -VV viruses, which had peaktiters of 10^(5.6)-10^(5.9) PFU/ml or lower in several separateexperiments, replicated less efficiently than the other viruses.

The PDK-13 virus-specific chimeras result in lower virus titersrecovered from transfected cells, relative to the 16007 virus-specificchimeras. Previous experiences with DEN-2/DEN-1 and DEN-2/DEN-4 chimerasindicate that chimeric viruses which exhibit crippled replication duringtransfection and later develop high virus titers after passage in cellculture often accrued unexpected mutations. Viruses of increasedreplicative ability may arise through selection of subpopulations ofvirus variants, resulting from incorporation errors during in vitrotranscription of cDNA. Chimeric viruses that replicate well intransfected cells were more genetically stable after passage in LLC-MK₂cells. Efficient replication with minimal passage in mammalian cellculture may be an important criterion of genetic stability andsuitability for an infectious clone-derived vaccine virus.

Viral growth curves in C6/36 cells were monitored following infection ofC6/36 cells at an approximate multiplicity of 0.001 PFU/ml (FIG. 3). Thethree DEN-2 backbone viruses, DEN-2-16681-P48, DEN-2-PDK53-E48 andDEN-2-PDK53-V48, replicated hie the original DEN-2 16681 virus and thetwo PDK-53 variants, respectively. Both DEN-2-PDK53-E48 andDEN-2-PDK53-V48 viruses replicated about 4000-fold less efficiently thanthe DEN-2-16681-P48, DEN-1 16007 and PDK-13 viruses in C6/36 cells.DEN-1 16007 and PDK-13 viruses replicated to high titers of 10^(8.7) and10^(8.4) PFU/ml, respectively. The chimeric DEN-2/1-PP virus replicatedto 10^(5.2) PFU/ml, which was equivalent to the peak titers of the twoDEN-2-PDK53 variants. Replication of chimeric DEN-2/1-EP and DEN-2/1-VPviruses was very inefficient in C6/36 cells. These viruses reached peaktiters of lower than 10² PFU/mL

Neurovirulence in Suckling Mice

Groups of newborn to one-day-old ICR mice (n=16) were inoculatedintracranially with 5000 PFU of virus. Wild-type DEN-2 16681 virus was100% fatal with average survival time at 14.1±1.6 days, while bothclone-derived DEN-2-PDK53-E48 and DEN-2-PDK53-V48 viruses failed to killany ICR mice. Unlike DEN-2 16681 virus, which typically kills 50% orgreater of challenged mice, the wild-type DEN-1 16007 virus caused onlya single fatality (21 days after challenge) in the ICR mice. The DEN-1PDK-13 virus did not kill any of the ICR mice. DEN-1 16007virus-infected ICR mice had significantly lower mean body weights(p<0.00003, Student's t test), relative to the control group inoculatedwith diluent, between 21-35 days after challenge. All of the ICR mousegroups challenged with five chimeric DEN-2/1 viruses (DEN-2/1-VV viruswas not tested) had lower mean weights (p<0.02) when compared to thecontrol group, but their mean weights were significantly greater(p<0.004) than the DEN-1 16007 group 28 days after infection. The meanbody weights of ICR mouse groups challenged with 10⁴ PFU of DEN-1 16007or PDK-13 virus were nearly identical to those of the ICR micechallenged with 5000 PFU of DEN-1 16007 virus between 7-35 days afterchallenge.

Immunogenicity of Chimeric DEN-2/1 Viruses in Mice

Outbred ICR Mice: To test the immunogenicity of the chimeric viruses,groups of 3-week-old ICR mice (n=8) were immunized intraperitoneallywith 10⁴ PFU of virus in Experiment 1 and Experiment 2 (Table 7). InExperiment 1, the mice were bled 20 days after primary immunization andthen boosted 2 days later. In Experiment 2, the mice were bled 41 daysafter primary immunization and boosted 2 days later. Table 7 shows thereciprocal, 50% plaque-reduction endpoint PRNT titers of the pooledserum samples from each immunized group. The range of individual titersfor the eight mice in many of the groups is also shown. In bothexperiments, the reciprocal titer of the pooled serum from 16007virus-immunized mice was 80 before boost and 2560 after boost. In bothexperiments, mice immunized with chimeric DEN-2/1-PP, -EP, or DEN-2/1-VPviruses had a pooled serum titer that was at least as high as those ofthe 16007 virus-immunized groups before (reciprocal titers of 40-160)and after (reciprocal titers of 2560-5210) boost. The immune responsesof the mice in these virus groups were nearly equivalent in bothExperiment 1 and Experiment 2.

The PDK-13 virus and all three of the chimeras containing the PDK-13structural genes induced minimal or low reciprocal PRNT titers of 10-20against DEN-1 16007 virus by 20 days after primary immunization inExperiment 1. A somewhat higher reciprocal PRNT titer of 40 was elicitedby each of these viruses by 41 days after immunization in Experiment 2.The development of neutralizing antibodies was slower and of lowermagnitude following immunization with PDK-13, DEN-2/1-PV, -EV, or -VVvirus than with 16007, DEN-2/1-PP, -EP, or -VP virus in these mice. Onemouse in the DEN-2/1-PV group in Experiment 1 and one mouse in each ofthe DEN-2/1-EV groups in both experiments failed to produce a detectablePRNT titer before boost. Boosted titers were also higher for the PDK-13,DEN-2/1-PV, -EV, and -VV-immunized groups in Experiment 2 (pooledreciprocal titers of 160-2560) than in Experiment 1 (pooled reciprocaltiters of 80). Except for the DEN-2/1-PV group in Experiment 2, theseboosted titers were lower than the boosted PRNT titers induced bywild-type 16007 virus and chimeric DEN-2/1-PP, -EP, and -VP virusescontaining the structural genes of the wild-type DEN-1 16007 virus(Table 7). The high PRNT titer obtained for the pooled serum of the miceboosted with DEN-2/1-PV virus resulted from two mouse sera which hadreciprocal titers of 2560 and 10,240. The remaining 6 mice in this grouphad reciprocal titers of 20 to 640, which were similar to the individualtiters of mice in the PDK-13, DEN-2/1-EV, and -VV groups. The PDK-13,DEN-2/1-PV, DEN-2/1-EV, and DEN-2/1-VV viruses appeared to be lessimmunogenic than the 16007, DEN-2/1-PP, -EP, and -VP viruses in theseoutbred mice. Pooled serum samples from mice immunized withDEN-2-1668′-P48, DEN-2-PDK-53-E48, or DEN-2-PDK-53-V48 virus did notcontain detectable cross neutralizing antibody against DEN-1 16007virus.

Inbred AG-129 Mice: To test the immunogenicity of the chimeric viruses,groups of 3.5-4.5-week-old inbred AG-129 mice were immunizedintraperitoneally with 10⁴ PFU of wild-type DEN-1 16007, Mahidolcandidate vaccine DEN-1 PDK-13, chimeric DEN-2/1-EP, or chimericDEN-2/1-VP virus. At 26 days after primary immunization, pooled serafrom mice immunized with chimeric DEN-2/1-EP or DEN-2/1-VP virus hadreciprocal 70% serum dilution-plaque reduction neutralizing antibodytiters (PRNT₇₀) of 80-160. These titers were equivalent (within 2-fold)of the neutralizing antibody response elicited by the candidate DEN-1PDK-13 vaccine, but lower than the 640 titer elicited by the wild-typeDEN-1 16007 virus. Control mice injected intraperitoneally withphosphate buffered saline had titers of less than 10. All mice werechallenged intraperitoneally with 10⁷ PFU of wild-type, virulent DEN-1virus, strain Mochizuki, at 28 days after primary immunization. Alleleven of the control, non-immunized mice died within 21 days (averagesurvival time of 11.5±4.2 days [mean±standard deviation]) afterchallenge, whereas all of the DEN-1 (16007, PDK-13, DEN-2/1-EP,DEN-2/1-VP) virus-immunized mice survived challenge with DEN-1 Mochizukivirus. At 34 days after challenge with DEN-1 Mochizuki, all of thevirus-immunized mice had reciprocal PRNT₇₀ titers of 1280-2560. Thechimeric DEN-2/1-EP and DEN-2/1-VP viruses were highly immunogenic andprotective for AG-129 mice.

Nucleotide Sequence Analyses of DEN-1 16007 and PDK-13 Viral Genomes

We sequenced the genomes of wild-type DEN-1 16007 virus (GenBankaccession number AF180817) and its PDK-13 vaccine derivative (accessionnumber AF180818). There were 14 nucleotide and 8 encoded amino aciddifferences between 16007 and PDK-13 viruses (Table 2). Silent mutationsoccurred at genome nucleotide positions 1567, 2695, 2782, 7330, and 9445in the E, NS1, NS4B, and NS5 genes. Unlike the candidate DEN-2 PDK-53vaccine virus, which has no amino acid mutations in the E protein, theDEN-1 PDK-13 virus had five amino acid mutations in E, including E-130Val-to-Ala, E-203 Glu-to-Lys, E-204 Arg-to-Lys, E-225 Ser-to-Leu, andE-447 Met-to-Val. Amino acid mutations in the nonstructural genesincluded NS3-182 Glu-to-Lys, NS3-510 Tyr-to-Phe, and NS4A-144Met-to-Val. The PDK-13 virus-specific E-477-Val was incorporated intoall of the chimeric constructs.

Immunization of Monkeys with Chimeric DEN-2/1 Viruses

The immunogenicity of the chimeric DEN-2/1-EP and DEN-2/1-VP viruses inadult crab-eating monkeys (Macaca fascicularis) was tested. Immunizationof monkeys was accomplished by means of subcutaneous injection with 10⁶PFU of chimeric DEN-2/1-EP or DEN-2/1-VP virus. Sera obtained from theimmunized monkeys were analyzed for the presence of viremia by directplaque assay of serum aliquots in LLC-MK₂ cell monolayers maintainedunder agarose overlay in 6-well plates, and by inoculation of serumaliquots into cultures of LLC-MK2 cells maintained in liquid medium. Noinfectious virus was identified in any of the monkey sera by either ofthese two classical assay methods. By these two virus assays, no viremiawas detectable following immunization with either chimeric DEN-2,1-EP orDEN-2/1-VP virus. Monkey sera were tested for DEN-1 virus-specificneutralizing antibodies. At 30 days after primary immunization, serafrom three individual monkeys immunized with chimeric DEN-2/1-EP virushad reciprocal 50% serum dilution-plaque reduction neutralizing antibodytiters (PRNT₅₀) of 80, 160 and 1280. Sera from three individual monkeysimmunized with chimeric DEN-2/1-VP had reciprocal PRNT₅₀ titers of 160,160 and 640. Monkeys injected with diluent as a control for theexperiment had reciprocal PRNT₅₀ titers of less than 10, as did all ofthe monkeys just prior to immunization. The chimeric DEN-2/1-EP and -VPviruses elicited DEN-1 virus-specific neutralizing antibodies innon-human primates.

Example 2 Construction of Chimeric DEN-2/3 Infectious Clones

An in vitro ligation strategy was used for the full genome-lengthDEN-2/3 infectious clones.

(i) 5′-end DEN-2/3 intermediate clones: pD2I/D3-P1-Asc andpD2I/D3-E1-Asc

Intermediate DEN-2 clones, pD2I-P and pD2I-E were used to subclone theDEN-3 16562 virus-specific cDNA fragment. The cDNA fragment containingthe prM-E genes of wild-type DEN-3 16562 virus was amplified by reversetranscriptase-polymerase chain reaction (RT-PCR) from DEN-3 viral RNAwith primers D3-435.Mlu: (5′-TGCTGGCCACTTAACTACGCGTGATGGAGAGCCGCGCA-3′(SEQ ID NO:33); underlined MluI site followed by DEN-3 virus sequencenear the 5′ end of the prM gene) and cD3-2394.Ngo:

(SEQ ID NO: 34) (5′-TGTAATGATGCCGGCCGCGATGCATGAAAATGA-3′;underlined NgoMIV site followed by complementary sequence near the 3′end of the E gene of DEN-3 virus). The MluI site contained a silentmutation for DEN-2 virus at amino acid prM-5-Thr. This position is Serin DEN-3 virus, but Thr in chimeric DEN-2/3 virus. The NgoMIV siteresulted in a Val-to-Ala substitution at E-482 of DEN-2 virus, and anIle-to-Ala substitution at E-480 of DEN-3 virus. The amplified fragmentwas cloned into the MluI-NgoMIV sites of the intermediate pD2I-P andpD2I-E clones. A restriction site, AscI, was introduced downstream ofthe NgoMIV site by site-directed mutagenesis to facilitate in vitroligation. This unique AscI site was only 16 nts downstream from theNgoMIV site and was excised prior to in vitro ligation of thefull-length DEN-2/3 clones. An additional mutagenesis at nt 1970(A-to-T) which changed amino acid E-345 from His to Leu, was introducedto permit derivation of viable chimeric viruses in LLC-MK₂ cells, asexplained below. These intermediate chimeric DEN-2/3 clones,pD2I/D3-P-Asc and pD2I/D3-E-Asc, were sequenced to verify the accuracyof the inserted DEN-3 virus-specific cDNA. A silent mutation wasincorporated at nt 552 (C-to-T) in both intermediate chimeric clones.3′-end DEN-2 intermediate clones: pD2-Pm̂b-Asc, pD2-Em̂Ab-Asc, andpD2-Vm̂b-Asc

Intermediate DEN-2 clones containing the truncated DEN-2 virus-specificcDNA sequence from nt 2203-10723 (3′-end) of DEN-2 16681, PDK53-E, orPDK-53-V virus were obtained by deletion of the 5′ end (including T7promoter sequence and DEN-2 nts 1-2202) of the virus specific cDNA inthe full-length clones, pD2-16681-P48, pD2-PDK53-E48, and pD2-PDK53-V48,respectively. An AscI site was also introduced at nt 2358 (22 ntsupstream of the NgoMIV site) to facilitate the in vitro ligation. ThisAscI site was excised prior to performing the in vitro ligation of thefull genome-length, chimeric DEN-2/3 infectious clones.

(iii) Full-length chimeric DEN-2/3 cDNA: DEN-2/3-PP1, DEN-2/3-EP1, andDEN-2/3-VPI

Three to ten mg of the 5′-end pD2I/D3 and 3′-end D2 intermediate cloneswere digested by AscI, treated with calf intestine phosphatase (CIP),and then digested with NgoMIV. The excised small AscI-NgoMIV fragmentswere removed by passing the digested DNA through Qiagen PCR-purificationspin columns. The large 5′- and 3′-end, linearized intermediate cloneswere then ligated together (5′:3′=1:3 molar ratio) to obtain fullgenome-length DEN-2/3-PP1 (pD2I/D3-P1-Asc ligated with pD2-Pm̂b-Asc),DEN-2/3-EP1 (pD2I/D3-E1-Asc ligated with pD2-Em̂b-Asc), and DEN-2,3-VP1(pD2I/D3-E1-Asc ligated with pD2-Em̂b-Asc). These ligated DNAs were thencut with XbaI to produce the linearized 3′-end of the viral cDNArequired for transcription of the recombinant viral RNA.

Recovery of Chimeric DEN-2/3 Viruses

Recombinant viral RNA was transcribed from XbaI-linearized cDNA andcapped with the cap analog m⁷ GpppA. LLC-MK₂ or BHK-21 cells (3-5×10⁶cells) were transfected by electroporation as described by Kinney et al.(J. Virol 230: 300-308 (1997)). Transfected cells were transferred to75-cm² flasks in DMEM medium containing 10% FBS. Viral proteinsexpressed in the transfected cells were analyzed by indirectimmunofluorescence assay (IFA). Virus-infected cells were fixed inice-cold acetone for 30 min. DEN-3 and DEN-2 virus-specific monoclonalantibodies 8A1 and 3H5, respectively, were used in the assay, andbinding was detected with fluorescein-labeled goat anti-mouse antibody.Viruses were harvested after 5 to 11 days, and were then passaged inLLC-MK₂ cells once to obtain working seeds. Viral genomes extracted fromthese seeds were sequenced to confirm the genotypes of the progenyviruses. An earlier strategy using 5′-end D2I/D3 intermediate clonescontaining authentic DEN-3 16562 prM-E genes resulted in mutations atseveral different positions in the genomes of the viruses recovered fromtransfected LLC-MK₂ or BHK-21 cells and passaged once in LLC-MK₂ cells.A mutation at nt 1970 from A to T, which changed amino acid E-345 fromHis to Leu, was found in seven of nine independently recoveredrecombinant viruses. It was obvious that the original DEN-2/3 chimericviruses were unstable in LLC-MK₂ and/or BHK-21 cells. The singlemutation at E-345 was the only mutation that occurred in the genomes ofthree recovered viruses, indicating that this mutation might help tostabilize the viruses in culture. We introduced this mutation in all theinfectious DEN-2/3 clones and recombinant viruses recovered from suchmutagenized clones proved to be stable in cell culture.

Example 3 Construction of Chimeric DEN-2/4 Infectious Clones

pD2-16681-P48, pD2-PDK53-E48, pD2-PDK53-V48 vectors

The three DEN-2 backbone vectors used for construction of the chimericDEN-2/4 clones were modified as described above. The DEN-2 infectiousclones have been previously reported in Kinney et al., 1997. ClonepD2-16681-P48 was modified from pD2/IC-30P-A to contain cloning sitesMluI and NgoMIV at nucleotide positions 451 and 2380, respectively. Thesame cloning sites were introduced into both DEN-2 PDK-53 virus-specificclones, pD2/IC-130Vx-4 and -130Vc-K, and the modified clones weredesignated as pD2-PDK53-E48 and pD2-PDK53-V48, respectively. Two cloningerrors were found in the original pD2/IC-130Vx-4 and -130Vc-K at nt-6665and nt-8840. These defects were corrected in pD2-PDK53-E48 and -V48. Theintroduced NgoMIV cloning site resulted in two nucleotide mutations (nt2381 and 2382; TG to CC), which encoded a Val-to-Ala substitution atE-482 of DEN-2 virus. The nucleotide changes introduced at the MluI sitewere silent for DEN-2 virus and chimeric DEN-2/4 viruses. The MluI site(near the C/prM junction) and NgoMIV site (close to E/NS1 junction) wereused to clone the prM-E genes of heterologous viruses.

Chimeric pDEN-2/4-PP1, -EP1, and -VPI

Two intermediate DEN-2 clones, pD2I-P and pD2I-E, were constructed bydeleting the HpaI (nt-2676) to XbaI (3′ terminus of viral genomic cDNA)fragments of pD2-16681-P48 and pD2-PDK53-E48, respectively. Theseintermediate clones were used to subclone DEN-4 1036 virus-specific cDNAfragments. The cDNA fragment containing the prM-E genes of DEN-4 1036virus was amplified by reverse transcriptase-polymerase chain reaction(RT-PCR) from DEN-4 viral RNA with primers D4-453Mlu:(5′-GGCGTTTCACTTGTCAACGCGTGATGGCGAACCCCTCA-3′ (SEQ ID NO:35); underlinedMluI site followed by sequence near the 5′ end of the DEN-41036 viralgenome) and cD4-2394.Ngo:

(SEQ ID NO: 36) (5′-AGTGATTCCGCCGGCAGCTATGCACGTCATAGCCAT-3′;underlined NgoMIV site followed by complementary sequence near the 3′end of the E gene of DEN-4 virus). Amplified fragments were cloned intothe MluI-NgoMIV sites of the intermediate pD2I-P and pD2I-E clones.Intermediate, chimeric DEN-2/4 clones were sequenced to verify theaccuracy of the inserted DEN-4 virus-specific cDNA. A silent mutationwas incorporated at nt 1401 (A-to-G) in both intermediate clones.Fragments excised from the DEN-2/IC-P48, -VE48, and -VV48 clones withNgoMIV and ScaI (downstream of DEN-2 cDNA sequence in these plasmids)were cloned into NgoMIV/ScaI-cut chimeric D2/4 intermediate clones toobtain the full-length chimeric D2/4-PP, -BP, and -VP clones. However,transcribed RNA from these chimeric clones only produced viable DEN-2/4chimeric viruses in transfected C6/36 cells, but not in transfectedLLC-MK₂ cells. After passaging the chimeric viruses in C6/36 cells onemore time to obtain higher titers, these viruses were passaged inLLC-MK₂ cells five times to obtain stable chimeric DEN-2/4 viruses whichreplicated efficiently in LLC-MK₂ cells. Titers of the virus seedsincreased from 200 PFU/ml at the first LLC-MK₂ cell passage to over 10⁶PFU/ml at the fifth LLC-MK₂ cell passage.

The genomes of viruses from the first, second, third and fifth LLC-MK₂cell passages of the chimeric DEN-2/4-PP viruses were sequenced. Fourmutations, DEN-2 virus-specific, C-100 (Arg-to-Ser), DEN-4virus-specific E-364 (Ala-to-Ala/Val mix), DEN-4 virus-specific E-447(Met-to-Leu) and DEN-2 virus-specific NS4B-239 (Ile-to-Leu) wereidentified (amino acid positions based on chimeric DEN-2/4 virussequences). The three mutations located in the structural genes (C andE) were introduced into the chimeric DEN-2/4-PP, -EP, and -VP infectiouscDNA clones to obtain DEN-2/4-PP1, -EP1, and -VP1 clones, respectively.All three of these chimeric DEN-2/4 clones produced viable, high-titeredchimeric viruses in LLC-MK₂ cells immediately after transfectionindicating that these three mutations helped the viruses to replicate inLLC-MK₂ cells. Chimeric DEN-2/4 clones were also mutagenized to containdifferent combinations of the four mutations to determine whichmutations are needed for replication efficiency of the chimeric DEN-2/4viruses in LLC-MK₂ cells. The DEN-4 E-447 (Met-to-Leu) mutation alone ortogether with the DEN-4 E-364 mutation, in combination with the DEN-2C-100 (Arg-to-Ser) mutation was adequate to allow derivation of DEN-2/4virus in LLC-MK₂ cells.

Recovery of Recombinant Viruses.

Recombinant plasmids pD2/4-PP1, -EPI, and -VP1 all were grown inEscherichia coli XL1-Blue cells. Recombinant viral RNA was transcribedand capped with the cap analog m⁷ GpppA from 200-400 ng ofXbaI-linearized cDNA, and transfected into 3-5.10⁶ LLC-MK₂ cells.Transfected cells were transferred to 75-cm² flasks in DMEM mediumcontaining 10% FBS. Viral proteins expressed in the transfected cellswere analyzed by indirect immunofluorescence assay (IFA). Virus-infectedcells were fixed in ice-cold acetone for 30 min DEN-4 and DEN-2virus-specific monoclonal antibodies 1H10 and 3H5, respectively, wereused in the assay, and binding was detected with fluorescein-labeledgoat anti-mouse antibody. Viruses were harvested after 8 to 10 days, andwere then passaged in LLC-MK₂ cells once to obtain working seeds. Thegenomes of all these three working seeds were fully sequenced, and allthe chimeric DEN-2/4 viral genomes contained the expected sequences. Thenucleotide and amino acid sequences for the DEN 2/4 chimera are providedherein at SEQ ID NO:13 and SEQ ID NO:14, respectively.

Example 4 Characterization of DEN-2/3 and DEN-2/4 Chimeric Viruses

The viable, infectious chimeric DEN-2/3 and DEN-2/4 viruses, whichexpress the prM/E gene region of wild-type DEN-3 16562 or wild-typeDEN-4 1036 virus, respectively, expressed appropriate DEN-3 or DEN-4virus-specific envelope protein (E) epitopes, as analyzed by indirectimmunofluorescence of virus-infected LLC-MK₂ cells with virus-specificanti-E monoclonal antibodies. These chimeric viruses, as well aschimeric DEN-2/1 virus, also expressed appropriate DEN serotype-specificneutralization epitopes when tested against standard polyvalent mouseascitic fluids or monoclonal antibodies in serum dilution-plaquereduction neutralization tests (Table 8). The chimeric DEN-2/1-EP,DEN-2/3-EP1, and DEN-2/4-EP1 viruses were neutralized by these standardDEN virus-specific antibodies to reciprocal PRNT₅₀ titers that were atleast as high as those that occurred for wild-type DEN-1, DEN-3, andDEN-4 viruses, respectively (Table 8). These neutralization dataindicated that the chimeric DEN-2/1, DEN-2/3, and DEN-2/4 virusesexpressed appropriate DEN serotype-specific neutralization epitopes ofDEN-1, DEN-3, and DEN-4 viruses, respectively.

Replication in LLC-MK2 Cells

The replication and temperature sensitivity of DEN-2/3 chimeras inLLC-MK2 cells was monitored as the replication and temperaturesensitivity of DEN-2/1 chimeras was examined in. Example 1. The chimericDEN-2/3-PP1, -EP1, and -VP1 viruses and chimeric DEN-2/4-PP1, -EP1, and-VP1 viruses, which expressed the prM/E gene region of DEN-3 16562 virusor DEN-4 1036 virus in the genetic background of DEN-2 16681 (PP1),DEN-2 PDK-53-E variant (-EP1), or the DEN-2 PDK-53-V variant (-VP1),respectively, all replicated to high peak titers of at least 10⁶-3PFU/ml in LLC-MK₂ cells. As defined in terms of the reduction of virustiters at 38.7° C. versus those at 37° C. (−, +, 2+, 3+ indicate titerreduction of less than or equal to 60%, 61-90%, 91-99%, >99%,respectively, calculated from at least 3 experiments). ChimericDEN-2/3-PP1 and DEN-2/4-PP1 viruses exhibited either borderlinetemperature sensitivity (DEN-2/3-PP1) or no temperature sensitivity(DEN-2/4-PP1). The chimeric DEN-2/3-EP1 and -VP1, as well as thechimeric DEN-2/4-EP1 and -VP1 viruses, all of which were constructed inthe genetic background of the DEN-2 PD-53-E or -V variant, retained thetemperature-sensitive phenotypes that were exhibited by the two PDK-53variant viruses (DEN-2-PDK53-E48 and -V48 viruses, respectively). Thechimeras constructed in the background of the PDK-53-V variant exhibiteda higher degree of temperature sensitivity than did those constructed inthe background of the PDK-53-E variant.

Plaque Sizes in LLC-MK2 Cells

The plaque size resulting from inoculation of LLC-MK2 cells placed underagarose overlay, used as a biological marker to determine attenuation asdescribed in Example 1, was also examined for the chimeric DEN-2/3 andDEN-2/4 viruses. Average plaque size in mm follows each virus or chimerain brackets: DEN-3-16562 (6.6), DEN-2/3-PP1 (5.4), DEN-2/3-EPI (4.5),DEN-2/3-VP1 (2.3), DEN-4-1036 (8.6), DEN-2/4-PP1 (3), DEN-2/4-EP1 (1.5),DEN-2/4-VP1 (1.2), DEN-2-16681-P48 (4.2), DEN-2-PDK53-E48 (2.5) andDEN-2-PDK-53-V48 (1.8).

The plaque sizes of the DEN-2/3-PP1 and DEN-2/4-PP1 viruses exhibitedmean plaque diameters that were larger than those of the DEN-2 16681 orPDK-53 virus (as described in Example 1), but smaller than those ofwild-type DEN-3 16562 and DEN-4 1036 viruses, respectively. Thisindicates that structural genes from the donor DEN-3 and DEN-4 virusesand capsid and/or nonstructural gene regions in the recipient geneticbackground of DEN-2 16681 virus both affected plaque size. The chimericDEN-2/3-EP1 and -VP1 and DEN-2/4-EP 1 and -VP1 viruses exhibitedsignificant reductions in plaque size, relative to wild-type DEN-3 16562and DEN-4 1036 viruses, respectively. The DEN-2 PDK-53background-specific effect on plaque size may result from synergisticinteraction of the mutations at the NS1-53 and NS3-250 loci of DEN-2PDK-53 virus. Consequently, -VP 1 chimeras exhibited greater reductionsin plaque size than did the EPI chimeras. The chimeric DEN-2/1, DEN-2/3,and DEN-2/4 viruses constructed in the genetic background of thecandidate DEN-2 PDK-53 vaccine virus retained the phenotype of decreasedplaque size as exhibited by DEN-2 PDK-53 virus.

Replication in C6/36 Cells

The ability of the viruses to replicate in mosquito C6/36 cell culture,used as a biological marker to determine attenuation as described inExample 1, was also examined for the chimeric DEN-2/3 and DEN-2/4viruses. Average peak titers in units of Log₁₀, PFU/ml follow each virusor chimera in brackets; D3-16562 (7.3), DEN-2/3-PP1 (7.6), DEN-2/3-EP1(5.2), DEN-2/3-VP1 (5.7), D4-1036 (8.7), DEN-2/4-PP1 (7.8), DEN-2/4-EP1(4.5), DEN-2/4-VP1 (4.4), DEN-2-16681-P48 (8.3), DEN-2-PDK53-E48 (5.4)and DEN-2-PDK-53-V48 (5).

Both of the Mahidol candidate DEN-2 PDK-53 variants exhibit decreasedreplication efficiency, relative to wild-type DEN-2 16681 virus, inmosquito C6/36 cell culture (DEN-2-PDK-53-E48 and -V48 versusDEN-2-16681-P48 virus). The decreased replication ability in C6/36 cellshas been attributed to the mutations at the two 5′-NC-57 and NS1-53 lociin the DEN-2 PDK-53 virus; consistent with this view, both variantsreplicated to equivalently reduced peak titers in these cells. Thiscrippled replication phenotype in C6/36 cells was retained in thechimeric DEN-2/3-EP1 and -VP1 and DEN-2/4-EP1 and -VP1 viruses, all ofwhich replicated to lower peak titers than did the wild-type DEN-3 16562or DEN-41036 virus, respectively. The chimeric DEN-2/3-PP1 andDEN-2/4-PP1 viruses, constructed in the genetic background of wild-typeDEN-2 16681 virus, replicated to essentially the same extent(DEN-2/3-PP1) as or somewhat lower (DEN-2/4-PP1) than the wild-typeDEN-3 16562 and DEN-4 1036 viruses, respectively. These results indicatethat the replication-crippling effect of the 5′-NC-57 and NS1-53 loci inthe DEN-2 PDK-53 virus-specific background was preserved in thosechimeric viruses that were constructed within the DEN-2 PDK-53 geneticbackground.

Neurovirulence for Newborn Mice

Newborn, outbred, white ICR mice (n=16 for each group) were challengedintracranially with 10⁴ PFU of wild-type DEN-3 and DEN-4 viruses,Mahidol candidate vaccine DEN-3 and DEN-4 viruses, chimeric DEN-2/3-PPI,-EPI, -VP1 viruses, and chimeric DEN-2/4-PP1, -EP1, and -VP1 viruses(Table 9). The wild-type DEN-3 16562 and DEN-4 1036 viruses, whichreliably caused 100% fatality in newborn mice, constituted a moresensitive model for attenuation of viral neurovirulence than does theDEN-2 16681 challenge model, which results in 50-100% fatality innewborn mice challenged with this virus. Interestingly, the Mahidolcandidate DEN-4 PDK-48 vaccine virus also resulted in 100% fatality inchallenged mice, although the average survival time of these mice wasabout two days longer than for mice challenged intracranially withwild-type DEN-4 1036 virus (Table 9). Like the DEN-2 PDK-53 vaccinevirus and both of its variant populations, the chimeric DEN-2/3-EP1 and-VP1 and chimeric DEN-2/4-EP1 and -VP1 viruses were attenuated (nofatalities) for newborn mice. This attenuation may be attributable, atleast in part, to the attenuated DEN-2 PDK-53 genetic background ofthese chimeric viruses, because the chimeric DEN-2/4-PP1 virus exhibitedsignificant neurovirulence (62.5% mortality) in these mice. This latterchimera was constructed in the genetic background of wild-type DEN-216681 virus.

Immunization of AG-129 Mice with Chimeric DEN-2/3 and DEN-2/4 Viruses

Inbred AG-129 mice were immunized intraperitoneally with 10⁵ PFU ofwild-type or Mahidol vaccine candidate DEN-3 or DEN-4 virus or with 10⁵PFU of chimeric DEN-2/3-EP1 or DEN-2,4-EP1 virus (Table 10). Thechimeric DEN-2/3-EP1 virus elicited reciprocal PRNT₇₀ titers of 80-320at 4-6 weeks after primary immunization. These titers were essentiallyequivalent to those elicited by the Mahidol DEN-3 vaccine virus (strainPGMK-30/FRhL-3). However, the chimeric DEN-2/4-EP1 virus elicitedneutralizing antibody titer of 20-40, which was lower than the 80-320titer elicited by the Mahidol vaccine candidate DEN-4 PDK-48 vaccinevirus. Nevertheless, both chimeric DEN-2/3 and DEN-2/4 viruses elicitedneutralizing antibody responses in AG-129 mice.

Example 5 Attenuation of a Dengue-2 Vaccine Virus Strain 16681 (PDK-53)Viruses and Cell Cultures

The parental DEN-2 16681 virus, several intermediate PDK passages(PDK-5, -10, -14, -35, and -45) of 16681 virus, recombinant 16681/PDK-53viruses, and the genetically characterized LLC-MK₂-1 passage of thecandidate PDK-53 vaccine virus were investigated.

Cell cultures of BHK-21 (clone 15), LLC-MK₂, Vero, and C6/36 were grownin Dulbecco's modified minimal essential medium (DMEM) supplemented with10% heat-inactivated (56° C. for 30 min) fetal bovine serum (FBS;HyClone Laboratories, Inc., Logan, Utah), 3.7 g/L of sodium bicarbonate(GIBCO-BRL, Life Technologies, Gaithersburg, Md.), 100 units/ml ofpenicillin G, and 100 mg/ml of streptomycin sulfate (GIBCO-BRL).

Plaque titrations were performed in confluent monolayers of Vero cellsin plastic 6-well plates as described previously (Miller et al., Am. J.Trop. Med. & Hyg. 35: 1302-1309 (1986)): A 200-μl inoculum of virus wasadsorbed for 1.5 h at 37° C., followed by the addition of 4 ml ofagarose overlay medium containing 1% SeaKem LE agarose (FMC BioProducts,Rockland, Md.) in nutrient medium (0.165% lactalbumin hydrolysate [DifcoLaboratories, Detroit, Mich.], 0.033% yeast extract [Difco], Earl'sbalanced salt solution, 25 mg/L of gentamicin sulfate [Bio Whittaker,Walkersville, Md.], 1.0 mg/L of amphotericin B [Fungizone®, E. It Squibb& Sons, Princeton, N.J.], and 2% FBS). Following incubation at 37° C.for 7 days, a second 2-ml agarose overlay containing 80 μg/ml of neutralred vital stain (GIBCO-BRL) was added.

Construction of recombinant DEN-2 16681/PDK-53 viruses During thegenetic validation of clone-derived DEN-2 viruses in the present study,two cDNA cloning errors were discovered, nt-6665 A-to-G (NS4A-97Tyr-to-Cys) and nt-8840 A-to-G (NS5-424 Glu-to-Gly), in the previouslyreported PDK-53 virus-specific pD2/IC-130Vc-K. (NS3-250-Val variant)clone (Kinney et al., Virology 230: 300-308 (1997)). These defects werecorrected in a newly derived PDK-53 virus-specific (NS3-250-Val variant)clone, pD2/IC-VV45R.

In preliminary studies, recombinant 16681/PDK-53 viruses containingPDK-53 virus-specific gene regions within the genetic background of16681 virus were used to investigate the genetic loci involved in theattenuation markers of PDK-53 virus. Analyses of these viruses indicatedthat the PDK-53 mutation at nt-57 in the 5′NC region and the amino acidmutations at NS1-53 (analyzed in a linked manner with the NS2A-181mutation) and NS3-250 were the determinants of the PDK-53 virus-specificphenotype. The prM-29 mutation has little effect on virulence. Basedupon sequence analysis and comparison, the 5′NC, NS1 and NS3 mutationswere subjected to further mutational analysis. The 5′NC mutationoccurred in a possible stem structure. The NS1 and NS3 mutations bothoccurred at loci conserved in some flaviviruses. 14 recombinantpD2/IC-16681/PDK-53 plasmids were constructed by exchanging cDNAfragments between pD2/IC-30P-A (16681 clone) and pD2/IC-VV45R (PDK-53clone) at restriction enzyme sites SstI (preceding the T7 promoter),SalI (nt-165), SphI (nt-1380), SpeI (nt-2370 and nt-3579), KpnI(nt-4493), XhoI (nt-5426), and XbaI (3′ end of the clone). Allrecombinant plasmids were grown in Escherichia coli, strain XLI-blue,and were linearized at the unique XbaI site engineered at the 3′terminus of the cDNA. BBK-21 cells were transfected with transcribedviral RNA by the method of Liljestrom et al. (J. Virology 63: 4107-4113(1991)).

The genotypes of the recombinant D2/IC-Px (where x=5, 1, and/or 3 toindicate the incorporation of the parental [P in the virus designation]16681 virus-specific 5TTC-57, NS1-53, and/or NS3-250 loci into thepD2/IC-VV45R [PDK-53] backbone) and D2/IC-Vx (where x indicates thereciprocal incorporation of the three candidate PDK-53 vaccine [V in thevirus designation] virus-specific loci within the pD2/IC-30P-A [16681]backbone) viruses are shown in Table 11. If the 5′NC, NS1, and NS3 lociare the primary determinants of the PDK-53 virus-specific phenotype,then the D2/IC-P5 and D2/IC-V13 viruses should be equivalent (cognate)because both viruses contain 5′NC-57-C, NS1-53-Asp, and NS3-250-Val. Thecognate virus pairs derived from reciprocal mutagenesis of the 16681 andPDK-53 virus-specific infectious clones are indicated in Table 11. Tofurther investigate the prM-29 locus, we moved the prM-29-Asp locus ofDEN-2 16681 virus into pD2/IC-VV45R and pD2/IC-P5 to derive recombinantD2/IC-Pp and -P5p viruses, respectively. Reciprocal recombinationsyielded D2/IC-Vp and -V5p viruses.

Each clone-derived virus (transfected BHK-21 seed) was propagated oncein LLC-MK₂ cells. The genotypes of all of the LLC-MK₂-1-passaged,recombinant 16681/PDK-53 viruses were confirmed by complete nucleotidesequence analyses of their genomes. Because all of the viruses had theexpected nucleotide sequences, we inferred that their cDNA clones werealso correct. All of the clone-derived viruses contained the 16681virus-specific nt-8571-C locus, which is the site of a silent mutationin PDK-53 virus. Direct sequencing of overlapping cDNA ampliconsgenerated from DEN-2 viral genomic RNA using reversetranscriptase-polymerase chain reaction (RT-PCR) was used to determinethe sequence of all but the termini of the cDNA. The sequences of the5′- and 3′-terminal 30 nucleotides of the genome were determined bydirect sequencing of the infectious clone cDNA in plasmid pBRUC-139. TheD2/IC-prefix is eliminated in the virus designations in Table 11 and thefollowing text.

Characterization of the Replication Phenotypes of Recombinant16681/PDK-53 Viruses.

Viruses were analyzed for plaque size, temperature sensitivity, andreplication in LLC-MK₂ and C6/36 cells. Plaque sizes were evaluatedafter 9 days of incubation under agarose in LLC-MK₂ cell monolayersgrown in 6-well plates. Viral growth curves were performed in 75-cm²flasks of LLC-MK₂ or C6/36 cells inoculated at multiplicity of infection(m.o.i.) of approximately 0.001 PFU/cell. After adsorption at 37° C. for2 h, 30 ml of DMEM medium (LLC-MK, cells) or overlay nutrient medium(C6/36 cells) containing penicillin/streptomycin and 5% FBS was added,and the cultures were incubated in 5% CO₂ at 37° C. or 29° C.,respectively. Aliquots of culture medium were removed at 48-h intervals,adjusted to 12.5% FBS, and stored at −80° C. prior to virus titration.

Temperature sensitivity assays were performed in LLC-MK₂ cells grown in75-cm² flasks. The cells were inoculated at a m.o.i. of about 0.001PFU/cell. After adsorption for 2 h at 37° C., 30 ml of DMEM mediumcontaining 5% FBS was added. One set of cultures was incubated for 8days at 37° C., the other at 38.7° C. The ratio of virus titer at 38.7°C. versus the titer at 37° C. was calculated.

Mouse Neurovirulence Assay

Litters of newborn, outbred white ICR mice were inoculatedintracranially with 10⁴ PFU of virus in a volume of 30 ml. Mice wereindividually weighed once a week and were observed for paralysis ordeath for 35 days.

Plaque Phenotypes of Recombinant 16681/PDK-53 Viruses

Mean diameters of virus plaques (n=12) at 9 days after infection underagarose overlay in LLC-MK₂ cells were measured (FIG. 4). The largestplaques (3.2-3.4 mean diameter) were produced by the wild-type 16681virus, its clone-derived 30P-A virus, and the recombinant P513 virus,which contained the 5′NC, NS1, and NS3 16681 virus-specific loci in theVV45R (PDK-53) virus genetic background (Table 11). These three16681-specific loci within the PDK-53 genetic background were sufficientto reconstitute the large plaque phenotype of the 16681 virus.

Individual PDK-53 virus-specific 5′-NC-57-T, NS1-53-Asp, and NS3-250-Valmutations incorporated into the 16681 genotype each resulted insignificantly (p<0.00003) decreased plaque sizes of 2.1-2.3 mm in V5,V1, and V3 viruses, respectively, versus those of 16681 virus. Theplaque phenotypes of P13 and P51 viruses were similar to those of theircognate V5 and V3 viruses (indicated by graph bars with identical solidor cross-hatching pattern in FIG. 4). The 28-ram plaque size of P53virus differed from 16681 viral plaques to a lesser degree (p<0.03) thandid the 2.1-mm plaques of its cognate V1 virus. The 1.1-1.6-mm plaquesof cognate virus pairs V13 (P5) (containing PDK-53 virus-specific NS1and NS3 loci), V53 (P1) (PDK-53 virus-specific 5′NC and NS3 loci), andV51 (P3) virus (PDK-53 virus-specific 5′NC and NS1 loci) wereessentially equivalent to the 1.3-mm plaques of PDK-53 virus. Theseresults indicate that all pairwise combinations of these three PDK-53virus-specific loci in the 16681 backbone generated a small-plaque virussimilar to PDK-53 virus. Although V51 virus produced a plaque phenotypethat was similar to that of PDK-53 virus, all three 16681 virus-specific5′NC-C, NS1-53-Gly, and NS3-250-Glu loci were required within the PDK-53background of P513 virus to reconstitute the plaque phenotype of 16681virus. The presence of all three PDK-53 loci in the 16681 backbonegenerated V513 virus, which had a smaller (p<0.001, Student's t test)plaque phenotype than that of either PDK-53 or VV45R virus. Thedifference between the plaque size of PDK-53 virus and that of VV45Rvirus was not significant.

Recombinant Vp virus, which contained the PDK-53 virus-specificprM-29-Val locus in the 16681 background, had a significantly (p<0.002)reduced plaque size of 2.6 mm. However, the 1.0-mm plaque phenotype ofPp virus (16681 prM-29-Asp locus in the PDK-53 background) wasessentially identical to the 1.1-mm and 1.3-mm plaques of VV45R andPDK-53 viruses (FIG. 4A), respectively. The prM-29 locus did not affectthe plaque phenotypes of P5p (1.0±0.0 mm) and V5p (2.0±0.4 mm) viruses,which produced plaques similar to those of P5 (1.1±0 3 mm) and V5(2.3±0.4 mm), respectively.

Replication of Recombinant 16681/PDK-53 Viruses in LLC-MK₂ and C6/36Cells

All of the viruses replicated well in LLC-MK₂ cells, reaching peaktiters of 10⁷³-10″ PFU/ml at 6-8 days after infection. PDK-53 and itsclone-derived VV45R virus replicated at a reduced rate during the firstfour days after infection, relative to the other viruses. To determinetemperature sensitivities, virus-infected LLC-MK₂ cells were incubatedat 37° C. or 38.7° C. in 2-5 experiments (FIG. 4B). Temperaturesensitivity scores were determined at 8 days after infection. All of theviruses exhibited some degree of temperature sensitivity under theseconditions. In individual experiments, wild-type 16681 virus showed75-80% titer reduction at 38.7° C. Virus V1 and cognate viruses P3(V51), which showed 84-86% average reductions in titer, were slightlymore temperature-sensitive than 16681 virus. However, only PDK-53,VV45R, V513, P5p, Pp, and the cognate recombinant viruses V13 and P5,all of which contained both the NS1-53-Asp and NS3-250-Val PDK-53virus-specific loci, reproducibly showed 90-97% average reduction intiter at 38.7° C.

The 16681 virus and its clone-derived 30P-A virus replicated to averagepeak titers of 10′6-10″ PFU/ml at 12 days after infection in twoindependent growth-curve experiments in C6/36 cells (FIG. 4C). Thereplication of PDK-53 virus (peak titer of 10″ PFU/ml) and itsclone-derived VV45R (peak titer of 10″ PFU/ml) virus was approximately15,000-fold less efficient in C6/36 cells. The 16681 virus-specific 5′NCand NS1 loci within the PDK-53 background of P51 virus fullyreconstituted the replication efficacy to that of wild-type 16681 virus.Conversely, the PDK-53 virus-specific 5′NC and NS1 loci within the 16681background of V51 virus were sufficient to establish the crippledreplication phenotype of PDK-53 virus. Recombinant cognate virus pairsV5 (P13) and V1 (P53), which contained the PDK-53 virus-specific 5′NCregion or NS1 locus, respectively, replicated to average peak titers of10^(5.9)-10^(6.7) PFU/ml. Although the average peak titer of V53 viruswas about 40-fold greater than that of V5 virus in C6/36 cells, the peaktiters of P513, P13, P53, V3, V13, V513, and P3 viruses were verysimilar to those of P51, P1, P5, 30P-A, V1, V51, and VV45R viruses,respectively. These data indicated that the NS3-250 locus had little orno observable effect on replication in C6/36 cells (FIG. 4C). Vp and16681 viruses had nearly equal average peak titers in C6/36 cells, asdid P5 and P5p viruses. Pp and V5p viruses produced average peak titersthat were slightly higher (8- and 40-fold, respectively) than those ofPDK-53 and V5 viruses, respectively. The prM-29 locus appeared to havelittle or no effect on viral replication in C6/36 cells.

Neurovirulence of Recombinant 16681/Pdk-53 Viruses in Newborn Mice

To investigate the neurovirulence of the recombinant viruses, twolitters of newborn white ICR mice, eight mice per litter, were infectedintracranially with 10⁴ PFU of virus. The DEN-2 16681 virus and itsclone-derived 30P-A virus cause 50%-100% mortality in these mice.Average survival times (AST) for mice succumbing to challenge with 16681or 30P-A virus ranged from 15.2 to 16.8 days in various experiments.Mice were weighed individually every 7 days after infection. A singlemouse died by day 1 after infection, presumably as a result ofinoculation trauma, in each of the P53 and Vp groups (Table 12). Thesetwo mice were excluded from the analyses. There were no fatalities andno weight loss in the control, diluent-inoculated group.

Three mouse neurovirulence phenotypes were observed (Table 12). Thefirst phenotype consisted of the mouse-virulent viruses DEN-2 16681,30P-A, P513, P51, V3, and Vp, which caused at least 50% mortality withAST of 13.2-17.0 days (Table 12). In two other independent experiments,the Vp virus caused 46.67% mortality with AST of 17.4±1.4 days (n=16)and 56.25% mortality with AST of 18.3±1.3 days (n=16). In an independentexperiment, P51 virus caused only 25% mortality with AST of 15.9±5.5days (n=16). A second phenotype consisted of the mouse-attenuatedPDK-53, VV45R, V513, Pp, and cognate V51 (P3) viruses, which caused nomortality, and the nearly attenuated V1, cognate V13 (P5), P5p, and V5pviruses, which killed only 1 of 16 mice (Table 12). The presence of thetwo PDK-53 virus-specific 5′NC-57-T and NS1-53-Asp loci within the 16681genetic background was sufficient to result in or maintain attenuationin cognate viruses V51 (P3). Except for P53 virus, all of the virusescontaining the PDK-53 virus-specific NS1-53-Asp locus were attenuated ornearly attenuated.

The third phenotype, that of intermediate virulence, characterizedcognate virus pairs V5 (P13) and V53 (P1), and P53 virus which caused18.75%-37.5% mouse mortality and significant weight loss (p<0.001,Student's t test, at 3 weeks after infection, relative todiluent-inoculated control mice) in mice that survived virus challenge.Viruses V5 (P13) and V53 (P1) contained the 5′NC-57-T, but not theNS1-53-Asp, locus of PDK-53 virus. V1 virus (6.25% mortality) was moreattenuated than V5 virus, which produced 18.75% mortality andsignificant weight loss in the survivors. Conversely, the 16681virus-specific 5′-NC-57-C locus caused little reversion to virulence inP5 virus (6.25% mortality), whereas the NS1-53-Gly moiety in P1 virusresulted in an intermediate level (37.5%) of mortality and significantweight loss in the survivors. Unlike the nearly attenuated cognate V1virus, P53 virus had an intermediate virulence phenotype. The NS1-53locus had a more significant effect on the virulence phenotype than didthe 5′-NC-57 locus.

The prM-29 locus showed no effect in P5p, Pp, and Vp viruses, relativeto P5, PDK-53, and 16681 viruses, respectively. The V5p virus, whichcontained both PDK-53 virus-specific 5′NC-57-T and prM-29-Val loci, wasnearly attenuated (Table 2). The NS3-250 locus did not appear tocontribute significantly to mouse neurovirulence phenotype in V3, P13,V53, V13, and P3 viruses, which exhibited phenotypes that wereequivalent to 16681, P1, V5, V1, and PDK-53 viruses, respectively. Thedifference in the level of mortality caused by P53 and P5 virusessuggested that the 16681 virus-specific NS3-250-Glu locus mightcontribute somewhat to the virulence phenotype within certain geneticcontexts.

Evolution of Mutations in the DEN-2 PDK-53 Vaccine Virus

Intermediate passages PDK-5, -10, -14, -35, and -45 of the 16681 viruswere analyzed to determine the accrual of the nine nucleotide mutationsin the PDK-53 vaccine strain. Amplicons were amplified directly fromgenomic mRNA extracted from the viral seed by RT/PCR. Automatedsequencing of small genomic regions, which contained the nine relevantloci, was performed by using appropriate primers. The nucleotideresidues identified at each of the nine loci for these viruses are shownin Table 13. The NS2A-181 Leu-to-Phe mutation and the silent mutationsat E-37, NS3-342, and NS5-334 appeared by passage PDK-5 and were thepredominant moieties by passage PDK-10 (NS2A-181), PDK-14 (E-373,NS3-342), or PDK-35 (NS5-334). Mutations 5′-NC-57 C-to-T, prM-29Asp-to-Val, NS1-53 Gly-to-Asp, and NS4A-75 Gly-to-Ala occurred bypassage PDK-35. The 5′-NC-57-T was predominant at passage PDK-35, whilethe other listed mutations became predominant by passage PDK-45. TheNS3-250 Glu-to-Val mutation appeared by passage PDK-45 and is not fullymutated to the virus-specific Val in the current PDK-53 vaccinecandidate (Table 13). Approximately 29% of the viral population in thePDK-53 vaccine contains NS3-250-Glu. The PDK-45 virus was geneticallyequivalent to the PDK-53 vaccine virus. In the present study, no attemptwas made to determine the relative proportions of the two nucleotides atthe mixed genetic loci shown in Table 13.

Example 6 Construction of Chimeric DEN-2/West Nile Clones and Virus

Genome-length, chimeric DEN-2/WN infectious cDNA clones containingstructural genes of WN virus within the genetic background of DEN-2virus were constructed using the in vitro ligation strategy used toderive the chimeric DEN-2/3 viruses described earlier.

In a first example, the prM-E encoding cDNA of the 5′-end subclone thatwas used to derive a chimeric DEN-2/3-PP1 virus clone (see Example 2)was replaced with the prM-E gene region of WN virus, strain NY 99 (NewYork 1999). The cDNA fragment containing the prM-E gene region of WNvirus was amplified by reverse transcriptase-polymerase chain reaction(RT-PCR) from WN virus-specific genomic RNA with forward primer WN-M(5′-GGAGCAGTTACCCTCTCTACGCGTCAAGGGAAGGTGATG-3′ (SEQ ID NO:37);underlined MluI site followed by WN virus sequence near the 5′ end ofthe prM gene) and reverse primer cWN-E(5′-GAAGAGCAGAACTCCGCCGGCTGCGAGAAACGTGA GAGCTATGG-3′ (SEQ ID NO:38);underlined NgoMIV site followed by sequence that was complementary tothe WN genomic sequence near the 3′ end of the E gene of WN virus). Theamplified prM-E cDNA fragment was cloned into the MluI-NgoMIV sites ofthe intermediate pD2I/D3-P1-AscI clones, exactly as was done during thederivation of chimeric DEN-2/3 virus (see Example 2, construction ofchimeric DEN-2/3 infectious clones), to replace the prM/E region of theDEN-3 16562 virus. In this chimeric DEN-2/WN cDNA, the prM splice siteencoded the amino acid sequence (shown as single-letter amino acidabbreviations, and written in amino-to-carboxyl order)SAGMIIMLIPTVMA-FHLTTROGKVMMTV (SEQ ID NO:39). The hyphen in thissequence indicates the polyprotein cleavage site located between thecapsid and prM genes; the amino-terminal sequence is DEN-2 virusspecific; the carboxyl-terminal sequence in bold, underlined, italicizedfont indicates the WN virus-specific sequence (QGKVMMTV) (SEQ ID NO:40)near the amino terminus of the prM protein encoded by this chimericconstruct. This intermediate DEN-2/WN subclone was ligated in vitro withthe 3′-end intermediate DEN-2 subclone, pD2-Pm̂b-Asc, by the sameprotocol described in example 2 (construction of chimeric DEN-2/3infectious clones) to produce the full genome-length, chimeric DEN-2/WNviral cDNA that was used to transcribe chimeric DEN-2/WN virus-specificgenomic RNA. Using the same protocol described in example 2, mammalianLLC-MK₂ or mosquito C6/36 cells were transfected with the transcribedchimeric DEN-2/WN RNA. This strategy, which involved using the same MluIrestriction enzyme splice site that was used to derive the chimericDEN-2/3 and DEN-2/4 viruses, failed to produce viable chimeric virusafter transfection of the transcribed chimeric DEN-2/WN RNA in LLC-MK₂cells, and resulted in only very low titers (<100 PFU/ml) in C6/36cells. This failure is probably due to significant gene sequencevariation between the carboxyl-terminal ends of the viral capsidproteins and between the amino-terminal ends of the prM protein of DEN-2and WN viruses. The carboxyl-terminal region of the flavivirus capsidprotein serves as a signal peptide sequence for the insertion of prMinto intracellular membranes (endoplasmic reticulum) during maturationand cleavage of the prM protein. This DEN-2/WN construct, whichcontained the capsid-carboxyl-terminal signal sequence, as well as theamino-terminal residues of the prM protein of the DEN-2 backbone,apparently did not permit appropriate maturation of the chimeric virus.

In a second example, the chimeric DEN-2/WN cDNA clone was modified so asto encode the carboxyl-terminal region of the capsid protein, as well asthe entire prM protein and most of the E protein, of WN virus. A uniqueSstII restriction site was introduced by site-directed mutagenesis nearthe 3′ terminus of the DEN-2 virus-specific capsid gene to serve as anew 5′ splice site for the WN capsid-carboxyl-end/prM/E gene region.This SstII site introduced two silent mutations in the DEN-2virus-specific sequence encoding the amino acid triad SAG (single-letterabbreviations). The appropriate gene region was amplified from WN viralRNA by using the forward primer WN-452.5AG(5′-AATTCAACGCGTACATCCGCGGGCACCGGAATTGCAGTCA TGATTGGCCTGATGGC-3′ (SEQ IDNO:41); underlined SstII site followed by WN virus-specific sequence)and the same reverse cWN-E primer that was utilized in the previousconstruct (as described above). This amplified cDNA was cloned to makethe intermediate subclone pDEN-2/WN-P-SA which contained cDNA encodingthe 5′ noncoding region and most of the capsid gene from DEN-2 16681virus and the carboxyl-terminal capsid, entire prM, and most of the Egene from WN virus, as well as a unique AscI site downstream of theNgoMIV site. In this chimeric DEN-2/WN cDNA, the prM splice site encodedthe amino acid sequence (shown as single-letter amino acidabbreviations, and written in amino-to-carboxyl order)SAGTGIAVMIGLIASVGA-VTLSNFOGKVMMTV (SEQ ID NO:42). The hyphen in thissequence indicates the polyprotein cleavage site located between thecapsid and prM genes; the amino-terminal sequence is DEN-2 virusspecific; the carboxyl-terminal sequence in bold, underlined, italicizedfont indicates the WN virus-specific sequence(TGIAVMIGLIASVGA-VTLSNFQGKVMMTV) (SEQ ID NO:43) encoded by this chimericconstruct. Following the in vitro ligation protocol of the chimericDEN-2/3 clones described for chimeric DEN-2/3 virus in example 2, the5′-end intermediate subclone pDEN-2/WN-P-SA was ligated to the 3′-endpD2-Pm̂b-Asc subclone to produce the full genome-length cDNA of thechimeric DEN-2/WN-PP1 virus for transcription of the chimeric RNA. Thechimeric DEN-2/WN-PP1 construct encoded the indicated WN structural generegion within the genetic background of the wild-type DEN-2 16681 virus.Both LLC-MK₂ and C6/36 cells were transfected with RNA transcribed fromthis in vitro-ligated, chimeric DEN-2/WN-PP1 clone. Viable, chimericDEN-2/WN-PP1 virus, at virus titers of 10³-10⁶ PFU/ml of culture medium,was successfully recovered from both transfected cell cultures.Nucleotide sequence analysis of the entire genome of the clone-derived,chimeric DEN-2/WN virus demonstrated the expected genomic sequence.These results demonstrate that the DEN-2 infectious clones of theinvention can be used to construct chimeric viruses that expressstructural genes of heterologous flaviviruses other than DEN-1, DEN-3,and DEN-4 viruses.

All of the patents, publications and other references mentioned hereinare hereby incorporated in their entirety by reference. Modificationsand variations of the present methods and compositions will be obviousto those skilled in the art from the foregoing detailed description.Such modifications and variations are intended to come within the scopeof the appended claims

TABLE 1 Viral Candidates for Flavivirus Chimeras ⁺ PrimaryMosquito-Borne Species Other Mosquito-Borne: Species Dengue-1 ** AlfuyDengue-3 ** Bagaza Dengue-4 ** Banzi ** Yellow fever ** Bouboui Japaneseencephalitis ** Bussuquara ** Murray Valley encephalitis ** Edge Hill **St. Louis encephalitis ** Ilheus West Nile ** Naranjal Kunjin * Israelturkey meningitis Jugra Kokobera Ntaya Rocio ** Sepik ** Spondweni **Stratford Tembusu Uganda S Usutu ** Wesselsbron ** Zika ** Tick-Borne:No Arthropod Vector Demonstrated Absettarov ** Apoi * Gadgets Gully AroaHanzalova ** Cacipacore Hypr ** Carey Island Kadam Cowbone Ridge KarshiDakarbat ** Kumlinge ** Entebbe bat Kyasanur Forest disease ** JutiapaLangat Koutango * Louping ill ** Modoc * Meaban Montana Myotis leukemiaOmsk hemorrhagic fever ** Negishi ** Powassan ** Phnom-Penh bat RoyalFarm Rio Bravo ** Russian spring Saboya summer encephalitis ** Sal ViejaSaumarez Reef San Perlita Tyuleniy Sokuluk * = Laboratory infectionreported ** = Natural and laboratory infection reported ⁺ = Listincludes the currently classified members of the Flavivirus genus in theInternational Catalogue of Arboviruses, Including Certain Other Virusesof Vertebrates, Nick Karabatsos, ed. The American Society of TropicalMedicine and Hygiene. 1985.

TABLE 2 Summary Of Nucleotide And Amino Acid Differences Between TheGenomes Of DEN-1 16007 Virus And Its Vaccine Derivative, Strain PDK-13Genome Poly- Nucleotide Nucleotide Amino Acid Protein protein Position16007 PDK-13 16007 PDK-13 Position Position 1323 T C Val Ala E-130 4101541) G A Glu Lys E-203 483 1543) A G 1545 G A Arg Lys E-204 484 1567 AG Gln Gln E-211 491 1608 C T Ser Leu E-225 505 2363 A G Met Val E-477757 2695 T C Asp Asp NS1-92 867 2782 C T Ala Ala NS1-121 896 5063 G AGlu Lys NS3-182 1657 6048 A T Tyr Phe NS3-510 1985 6806 A G Met ValNS4A-144 2238 7330 A G Gln Gln NS4B-168 2412 9445 C T Ser Ser NS5-6243117

TABLE 3 Summary Of Nucleotide And Amino Acid Sequence DifferencesBetween The Genomes Of DEN-2 16681 Virus And Its Vaccine Derivative,Strain PDK-53 Genome Poly- Nucleotide Nucleotide Amino Acid Proteinprotein Position 16681 PDK-53 16681 PDK-53 Position Position  57 CT^([a]) — —  524 A T Asp Val prM-29 2055 C T Phe Phe E-373 653 2579 G AGly Asp NS1-53 828 4018 C T Leu Phe NS2A-181 1308 5270 A T/A GluGlu/Val^([b]) NS3-250 1725 5547 T C Arg Arg NS3-342 1817 6599 G C GlyAla NS4A-75 2168 8571 C T Val Val NS5-334 2825 ^([a])5′ noncodingregion. ^([b])The PDK-53 vaccine contains two genetic variants atnt-5270.

TABLE 4 Summary Of Nucleotide And Amino Acid Differences Between TheGenomes Of DEN-3 16562 Virus And Its Vaccine Derivative, StrainPGMK-30/Frhl-3 Genome Nucleotide Amino Acid Poly- Nucleotide PGMK-30PGMK-30/ Protein protein Position 16562 FRhL-3 16562 FRhL-3 PositionPosition  550 C T Ala Ala prM-38 152 1521 C/T C Ser/Leu^(a) Ser E-196476 1813 G A Lys Lys E-293 573 1838 A G Ser Gly E-302 582 1913 G A GluLys E-327 617 2140 C T Ala Ala E-402 682 3725 T C Phe Leu NS2A-86 12114781 C A Gln Lys NS3-90 1563 ^(a)Two significant genetic variants werelocated at nt-1521.

TABLE 5 Summary Of Nucleotide And Amino Acid Differences Between TheGenomes Of DEN-4 1036 Virus And Its Vaccine Derivative, Strain PDK-48Genome Poly- Nucleotide Nucleotide Amino Acid Protein protein Position1036 PDK-48 1036 PDK-48 Position Position 1211 T C Ile Ile E-91 370 1971G A Glu Lys E-345 624 3182 G C Gln His NS1-253 1027 6660 C T Leu PheNS4A-95 2187 6957 A A/T Ile Ile/Phe NS4B-44 2286 7162 T C Leu SerNS4B-112 2354 7546 C C/T Ala Ala/Val NS4B-240 2366 7623 G T/G AspTyr/Asp NS5-21 2508

TABLE 6 Summary Of Non-Silent Mutations Between The Genomes Of TheParent- Vaccine Strains Of DEN-1, DEN-2, DEN-3, And DEN-4 Viruses GenomeNucleotide Position DEN-1 DEN-2 DEN-3 DEN-4  57 5′NC-57 c-t  524 prM-29D-V 1323 E-130 V-A 1521 E-196 S/L-S 1541) E-203 E-K 1543) 1545 E-204 R-K1608 E-225 S-L 1838 E-302 S-G 1913 E-327 E-K 1971 E-345 E-K 2363 E-477M-V 2579 NS1-53 G-D 3182 NS1-253 Q-H 3725 NS2A-86 F-L 4018 NS2A-181 L-F4781 NS3-90 Q-K 5063 NS3-182 E-K 5270 NS3-250 E-V/E 6048 NS3-510 Y-F6599 NS4A-75 G-A 6660 NS4A-95 L-F 6806 NS4A-144 M-V 6957 NS4B-44 I-I/F7162 NS4B-112 L-S 7546 NS4B-240 A-A/V 7623 NS5-21 D-D/Y

TABLE 7 Immunogenicity of viruses in mice. Plaquereduction-neutralization titer^(a) against DEN-1 16007 virus Experiment1^(b) Experiment 2^(b) Primary Boost Primary Boost Immunizing Pooledsera Pooled sera Pooled sera Pooled sera virus (Range) (Range) (Range)(Range) DEN-1 16007 80 (20-80) 2560 (80-20480) 80 (20-160) 2560(160-5120) D2/1-PP 80^(c) 5120^(c)  40 (10-160) 5120 (160-10240) D2/1-EP80 (20-320) 10240 (640-20480) 160 (20-320) 5120 (2560-≧10240) D2/1-VP 40(10-160) 2560 (160-5120) 80 (10-320) 5120 (40-≧10240) DEN-1 PDK-13 10(10-40) 80 ^(e) 40 (20-80) 320 (20-640) D2/1-PV 10 (<10^(d)-20) 80^(c)40 (10-80) 2560 (20-≧10240) D2/1-EV 20 (<10^(d)-20) 80^(c) 40(<10^(d)-40) 160 (10-320) D2/1-VV 10 (10-40) 80 ^(e) 40 (10-160) 160(20-640) ^(a)Titers are the reciprocal dilution yielding at least 50%plaque reduction. ^(b)3-week-old outbred ICR mice were immunizedintraperitoneally with 10⁴ PFU of virus, and were boosted with the samevirus dose 3 weeks later in experiment 1, or 6 weeks later in experiment2. Primary = serum taken 20 days (experiment 1) or 41 days (experiment2) after primary immunization. Boost = serum taken 21 days after boostin both experiments. ^(c)Individual titers were not determined. ^(d)Onlyone mouse serum titer was less than 10 in these groups. ^(e)Bold titersindicate the pooled sera titers were 4 fold higher than the titerscalculated with 70% plaque reduction. All other pooled titers wereeither no different from or two fold higher than 70% plaque reductiontiters.

TABLE 8 Neutralization of chimeric DEN viruses by standard antibodiesAntibody Virus D1-AF^(a) D2-AF^(a) D2-H5^(b) D3-AF^(a) D3-8A1^(b)D4-AF^(a) DEN-1 16007^(c) 1280^(d)   40 20 40 <20 20 DEN-2/1-EP^(e)2560  ; ; ; ; ; DEN-2 16681^(c) 80 2560>   40960 40 <20 40 DEN-316562^(c) 80 160  40 1280 5120 40 DEN-2/3-EP1^(e) ; ; ; 2560 20480 ;DEN-4 1036^(c) 20 40 <20 80 <20 1280 DEN-2/4-EP1^(e) ; ; ; ; ; 2560^(a)AF = DEN-1 (D), DEN-2 (D2), DEN-3 (D3), and DEN-4 (D4)virus-specific mouse ascitic fluids (polyvalent antisera). ^(b)DEN-2 andDEN-3 virus-specific, envelope glycoprotein-specific (E-specific)monoclonal antibodies D2-H5 and D3-8A1, respectively. ^(c)Wild-type DENvirus. ^(d)Reciprocal serum dilution-plaque reduction neutralizationtiters (50% endpoint) are shown; ; = not tested ^(e)Chimeric DEN-2/1-EP,DEN-2/3-EP1, or DEN-2/4-EP1 virus expressing structural genes of DEN-116007, DEN-3 16562, or DEN-4 1036 virus, respectively.

TABLE 9 Neurovirulence of chimeric DEN-2/3 and DEN- 2/4 viruses innewborn white ICR mice Challenge Percent AST (SD) Virus^(a)Mortality^(b) (days)^(c) DEN-3 16562^(d) 100.0 14.1 (2.1) DEN-3P30/FRhl-3^(e) 15.0 16.2 (3.2) DEN-2/3-PP1^(f) 32.5 19.0 (2.1)DEN-2/3-EP1^(f) 0.0 — DEN-2/3-VP1^(f) 0.0 — DEN-4 1036^(d) 100.0  8.6(0.6) DEN-4 PDK-48^(e) 100.0 10.7 (1.5) DEN-2/4-PP1^(f) 62.5 17.8 (2.8)DEN-2/4-EP1^(f) 0.0 — DEN-2/4-VP1^(f) 0.0 — DEN-2 16681^(d) 87.5 15.2(2.6) DEN-2 PDK53-E48^(e) 0.0 — DEN-2 PDK53-V48^(e) 0.0 — Diluent 0.0 —^(a)Newborn mice were challenged intracranially with 10⁴ PFU of virus.^(b){Number of fatalities/total number (n = 16) of mice challenged} ×100. ^(c)Average survival time (AST) and standard deviation (SD); — =not applicable, becausE there were no fatalities in this group.^(d)Wild-type DEN serotype virus ^(e)Mahidol candidate DEN vaccinevirus: DEN-3 = PGMK-30/FRhL-3 (P30/FRhL-3); DEN-4 = PDK-48; DEN-2 =PDK-53-E variant (NS3-250-Glu locus) and PDK-53-V variant (NS3-250-Vallocus). Both PDK-53-E and -V variant viruses were derived frominfectious cDNA clones of the variants. ^(f)Chimeric DEN-2/3 or DEN-2/4virus. The prM/E gene region of DEN-3 16562 virus or DEN-4 1036 viruswas expressed in the genetic background of wild-type DEN-2 16681 virus(PP1 viruses), Mahidol candidate vaccine DEN-2 PDK-53-E variant (EP1viruses), or Mahidol candidate vaccine DEN-2 PDK-53-V variant (VP1viruses).

TABLE 10 Protective efficacy of chimeric DEN-2/1 viruses in AG-129mice^(a) Immunizing Virus 4 weeks 6 weeks DEN-3 16562^(b) 320^(c)   640DEN-3 P30/FRhL-3^(d) 160  320 DEN-2/3-EP1^(e) 80 320 DEN-4 1036^(b) 160 1280 DEN-4 PDK-48^(d) 80 320 DEN-2/4-EP1^(e) 20 40 Reciprocalneutralizing antibody titer against appropriate homologous DEN-3 orDEN-4 virus at 4 or 6 weeks after immunization ^(a)AG-129 mice are aninbred strain that lack receptors for interferon alpha/beta andinterferon gamma. Mice, 3.5-4.5 weeks in age, were immunizedintraperitoneally with 10⁵ PFU of virus. ^(b)Wild-type DEN virus.^(c)Reciprocal dilution of pooled serum that neutralized 70% or greaterof the input wild-type DEN-3 16562 virus that was used to test sera frommice immunized with DEN-3 or chimeric DEN-2/3-EP1 virus, or the inputwild-type DEN-4 1036 virus that was used to test sera from miceimmunized with DEN-4 or chimeric DEN-2/4-EP1 virus. ^(d)Mahidolcandidate vaccine virus, DEN-3 PGMK-30/FRhL-3 (P30/FRhL-3), DEN-4 PD-48.^(e)Chimeric DEN-2/3-EP1 or DEN-2/4-EP1 expressing the prM/E gene regionof DEN-3 16562 or DEN-4 1036 virus, respectively.

TABLE 11 Genotypes of recombinant DEN-2 16681/PDK-53 virusesClone-derived Dengue-2 16681 determinants in PDK-53 background^(a) virus(cognate)^(b) 5′NC-57 prM-29 NS1-53 NS2A-181 NS3-250 NS4A-75 DEN-2PDK-53 t V D F V A VV45R (V513) . . . . . . P5 (V13) c . . . . . P1(V53) . . G . . . P3 (V51) . . . . E . P51 (V3) c . G . . . P53 (V1) c .. . E . P13 (V5) . . G . E . P513 (30P-A) c . G . E . Dengue-2 PDK-53determinants in 16681 background^(c) 5′NC-57 prM-29 NS1-53 NS2A-181NS3-250 NS4A-75 DEN-2 16681 c D G L E G 30P-A (P513) . . . . . . V5(P13) t . . . . . V1 (P53) . . D . . . V3 (P51) . . . . V . V51 (P3) t .D . . . V53 (P1) t . . . V . V13 (P5) . . D . V . V513 (VV45R) t . D . V. ^(a)The genome of the candidate dengue-2 PDK-53 vaccine virus differsfrom that of its 16681 parent at nine nucleotide loci, including threesilent mutations (not shown), a mutation at genome nucleotide position57 in the 5′ noncoding region (5′NC-57; lower case letters), and fivenucleotides encoding amino acid mutations (upper case, single-letterabbreviations) at viral polypeptide positions premembrane (prM)-29,nonstructural protein 1 (NS1)-53, NS2A-181, NS3-250, and NS4A-75 (32).Genetic loci from the parental 16681 virus were engineered into the cDNAbackground of the PDK-53 virus-specific infectious clone, pD2/IC-VV45R.Dots indicate sequence identity with PDK-53 virus (NS3-250-Val variant).The candidate PDK-53 vaccine also contains a genetic variant that hasGlu at NS3-250 (32). ^(b)The genotypes of wild-type DEN-2 16681 virus,its attenuated vaccine derivative, DEN-2 PDK-53 virus, infectiousclone-derived VV45R (genetically equivalent to the PDK-53 NS3-250- Valvariant) and 30P-A (equivalent to wild-type 16681) viruses, andrecombinant 16681/PDK-53 viruses are shown. The numerical designationsfor recombinant Px and Vx viruses (where x = 5′NC, NS1, and/or NS3 loci)indicate parental (P in virus designation) 16681 virus-specific lociengineered into the PDK-53 virus-specific infectious cDNA clone (topseries) or reciprocal candidate PDK-53 vaccine (V in virus designation)virus-specific loci engineered into the 16681 clone (bottom series),respectively. P5 and V13 are cognate viruses, assuming that the PDK-53virus-specific phenotype is determined predominantly by the 5′NC-57,NS1-53, and NS3-250 loci. Both P5 and V13 viruses contain the 5′NC-57-c,NS1-53-Asp, and NS3-250- Val loci within the genetic backgrounds ofPDK-53 and 16681 viruses, respectively. ^(c)Genetic loci from PDK-53virus were engineered into the cDNA background of the 16681virus-specific infectious clone, pD2/IC-30P-A. Dots indicate sequenceidentity with 16681 virus.

TABLE 12 Neurovirulence of DEN-2 16681, PDK-53, and recombinant16681/PDK-53 viruses in newborn white ICR mice Mouse challenge^(a) Virusgenotype^(b) Mortality AST (SD) 5′-NC prM NS1 NS2A NS3 NS4A Virus(cognate)^(c) (%) (days) 57 29 53 181 250 75 DEN-2 16681 68.75 15.2(1.2) c D G L E G 30P-A (P513) 81.25 14.6 (2.3) . . . . . . P513 (16681)100.0 13.2 (1.6) . V . F . A P51 (V3) 50.0^(d) 15.9 (5.5) . V . F V A P1(V53) 37.5^(d) 19.0 (4.2) t V . F V A P13 (V5) 37.5^(d) 13.5 (2.1) t V .F . A P53 (V1) 20.0^(d) 17.0 (7.8) . V D F . A P5p (V13) 6.25 15.0 . . DF V A P5 (V13) 6.25 27.0 . V D F V A Pp (PDK-53) 0 —^(e) t . D F V A P3(V51) 0 — t V D F . A V3 (P51) 75.0^(d) 16.4 (3.2) . . . . V . Vp(16681) 87.5 17.0 (0.9) . V . . . . V53 (P1) 18.75^(d) 21.3 (6.1) t . .. V . V5 (P13) 18.75^(d) 21.7 (4.2) t . . . . . V5p (P13) 6.25 20.0 t V. . . . V13 (P5) 6.25 17.0 . . D . V . V1 (P53) 6.25 22.0 . . D . . .V51 (P3) 0 — t . D . . . V513 (PDK-53) 0 — t . D . V . VV45R (V513) 0 —t V D F V A DEN-2 PDK-53 0 — t V D F V A ^(a)Percent mortality andaverage survival time (AST) ± standard deviation (SD) of newborn,outbred white ICR mice challenged intracranially with 10⁴ PFU of virus.Sixteen mice per group, except for the P53 and Vp groups in which asingle mouse died by day 1 after infection, presumably as a result ofinoculation trauma. These two mice were excluded from the study. ^(b)Seetext for explanation of virus genotypes. Solid dots indicate sequenceidentity with 16681 virus. ^(c)See text for explanation of virus andcognate virus designations. ^(d)Mean body weight of surviving mice wassignificantly lower (p < 0.001, Student's t test) than that ofdiluent-inoculated control mice (not shown) at 3 weeks after infection.^(e)Average survival time is not applicable because mere was nomortality in this mouse group.

TABLE 13 Evolution of DEN-2 virus, vaccine strain PDK-53, during passageof the parental 16681 strain in primary dog kidney (PDK) cells Genomenucleotide position^(a)/Translated polypeptide position^(b)/Encodedamino acids^(c) 524 2055 2579 4018 5270 5547 6599 8571 prM-29^(b) E-373NS1-53 NS2A-181 NS3-250 NS3-342 NS4A-75 NS5-334 Virus 57^(n) D - V^(c)F - F G - D L - F E - V R - R G - A V - V 16681 C^(d) A C G C A T G CPDK-5 C A C/T^(e) G T/C A T/C G C/T PDK-10 C A T/C G T A C/T G T/CPDK-14 C A T G T A C G T/C PDK-35 T T/A T A/G T A C C/G T PDK-45 T T T AT A/T C C T PDK-53 T T T A T A/T C C T ^(a)Genome nucleotide positionsof the nine nucleotide sequence differences between 16681 and PDK-53viruses. Nucleotide position 57 lies within the 5′ noncoding region ofthe viral genome. ^(b)Protein designations are as follows: prM =premembrane protein; E = envelope glycoprotein; NS = nonstructuralprotein. ^(c)The virus-specific amino acid residues (16681-PDK-53) areshown for each amino acid position. ^(d)A, C, G, and T (cDNA sense)nucleotides are indicated. ^(e)Two genetic populations were identifiedfor this locus in the virus. The order of the two nucleotides reflectsrelative peak heights of the nucleotide signals in sequencechromatograms.

TABLE 14 Conservation of DEN-2 PDK-53 phenotypic attenuation markers inchimeric DEN viruses. DEN virus Attenuation phenotype DEN-2^(a) DEN-2/1DEN-2/3 DEN-2/4 Attenuation in mice + + + + Decreased replicationin + + + + C6/36 cells Small plaques in LLC-MK₂ + + + + cellsTemperature sensitivity in + + + + LLC-MK₂ cells ^(a)Mahidol candidateDEN-2 PDK-53 vaccine virus. The chimeric DEN-2/1, DEN-2/3, and DEN-24viruses were constructed in the DEN-2 PDK-53 genetic background,NS3-250-Val or -Glu variant. The phenotypes shown for chimeric virusesare representative of chimeric viruses constructed in the background ofeither variant of DEN-2 PDK-53 virus.

TABLE 15 Diagnostic Genetic Probes and Amplimers for the CandidateMahidol DEN-1, DEN-2, DEN-3, and DEN-4 Vaccine Viruses. Genetic LocusPrimer Designation SEQ ID NO: D1V-1323 Probe pcD1V-1308 44 F fD1V-129845 R rD1V-1347 46 D1V-1541, 1543, 1545 Probe pcD1V-1530 47 F fD1V-151948 R rD1V-1567 43 D1V-1567 Probe pD1V-1580 50 F fD1V-1545 51 R rD1V-160852 D1V-1608 Probe pcD1V-1595 53 F fD1V-1567 54 R rD1V-1626 55 D1V-2363Probe pD1V-2374 56 F fD1V-2334 57 R rD1V-2386 58 D1V-2695 ProbepcD1V-2686 59 F fD1V-2660 60 R rD1V-2735 61 D1V-2782 Probe pD1V-2767 62F fD1V-2752 63 R rD1V-2801 64 D1V-5063 Probe pcD1V-5052 65 F fD1V-504066 R rD1V-5095 67 D1V-6048 Probe pD1V-6059 68 F fD1V-6030 69 R rD1V-606970 D1V-6806 Probe pcD1V-6793 71 F fD1V-6780 72 R rD1V-6825 73 D1V-7330Probe pD1V-7343 74 F fD1V-7310 75 R rD1V-7351 76 D1V-9445 ProbepcD1V-9433 77 F fD1V-9419 78 R rD1V-9485 79 D2V-57 Probe pD2V-69 80 FfD2V-32 81 R rD2V-81 82 D2V-524 Probe pcD2V-513 83 F fD2V-492 84 RrD2V-551 85 D2V-2055 Probe pD2V-2067 86 F fD2V-2025 87 R rD2V-2080 88D2V-2579 Probe pcD2V-2568 89 F fD2V-2535 90 R rD2V-2603 91 D2V-4018Probe pD2V-4030 92 F fD2V-3993 93 R rD2V-4043 94 D2V-5270-E D2V-5270-VProbeE pD2VE-5279 95 ProbeV pD2VV-5279 96 F fD2V-5243 97 R rD2V-5295 98D2V-5547 Probe pD2V-5558 99 F fD2V-5521 100 R rD2V-5589 101 D2V-6599Probe pcD2V-6588 102 F fD2V-6569 103 R rD2V-6625 104 D2V-8571 ProbepD2V-8582 105 F fD2V-8535 106 R rD2V-8594 107 D3V-550 Probe pD3V-562 108F fD3V-525 109 R rD3V-575 110 D3V-1521 Probe pD3V-1533 111 F fD3V-1497112 R rD3V-1541 113 D3V-1813 Probe pcD3V-1804 114 F fD3V-1767 115 RrD3V-1838 116 D3V-1838 Probe pcD3V-1827 117 F fD3V-1813 118 R rD3V-1913119 D3V-1913 Probe pcD3V-1903 120 F fD3V-1891 121 R rD3V-1967 122D3V-2140 Probe pcD3V-2127 123 F fD3V-2116 124 R rD3V-2188 125 D3V-3725Probe pD3V-3738 126 F fD3V-3698 127 R rD3V-3745 128 D3V-4781 ProbepcD3V-4772 129 F fD3V-4762 130 R rD3V-4801 131 D4V-1211 (T-C) ProbepD4V-1222 132 F fD4V-1191 133 R rD4V-1250 134 D4V-1971 (G-A) ProbepcD4V-1957 135 F fD4V-1943 136 R rD4V-2010 137 D4V-3182 (G-C) ProbepD4V-3193 138 F fD4V-3154 139 R rD4V-3227 140 D4V-6660 (C-T) ProbepcD4V-6648 141 F fD4V-6638 142 R rD4V-6688 143 D4V-7162 (T-C) ProbepD4V-7174 144 F fD4V-7141 145 R rD4V-7188 146 Genetic Locus: D1V-1323 =Mutated genetic locus for candidate DEN-1 PDK-13 vaccine (V) virus atgenome nucleotide position 1323. Primer designations: p = TaqMan probesequence, mRNA-sense pc = TaqMan probe sequence, complementary-sense f =forward amplimer, mRNA-sense r = reverse amplimer, complementary-senseSEQ ID = probe or primer sequence.

TABLE 16 SEQ ID NO: for TaqMan probes and amplimers. SEQ ID NO: 445′-TTCATATTGAGCTATCTTTCCTTCTA-3′ 45 5′-GTGTGCCAAGTTTAAGTGTG-3′ 465′-TGGACGGTGACTATCACTG-3′ 47 5′-AAGCCATGATTTCTTTTTCATTGTCA-3′ 485′-CAGGGCTAGATTTTAACGAG-3′ 49 5′-AGTGGTAAGTCTAGAAACCAC-3′ 505′-TCCACAAACAGTGGTTTCTAGACT-3′ 51 5′-TGTTGCTGACAATGAAAAAGAA-3′ 525′-TCCAAGTCTCTTGGGATGTTA-3′ 53 5′-TGGGATGTTAAAGCCCCAGAGGT-3′ 545′-TCATGGCTTGTCCACAAACAG-3′ 55 5′-CCAGTAAATCTTGTCTGTTCC-3′ 565′-CGTCCCTTTCGGTGATGTGCATC-3′ 57 5′-ATTCTGCTGACATGGCTAGG-3′ 585′-TCCTAGGTACAGTGTGACC-3′ 59 5′-AGATTCCACTAACGTCTCCCACG-3′ 605′-AATTGAACCACATCCTACTTG-3′ 61 5′-TTGTGTTCCATGGGTTGTGG-3′ 625′-TATGATTTTAGCTTTTCCCCAGCTT-3′ 63 5′-GCCACAACCCATGGAACAC-3′ 645′-ATGAAGGTGGTGTTCTGTAC-3′ 65 5′-TCCTAAACACCTTGTCCTCAATCT-3′ 665′-GCTAAGGCATCACAAGAAGG-3′ 67 5′-CGATCCTGGATGTAGGTCC-3′ 685′-CAGCCCTCTTTGAGCCGGAGA-3′ 69 5′-AATATAAACACACCAGAAGG-3′ 705′-TCCCCGTCTATAGCTGCAC-3′ 71 5′-TGTCAATATCACGAATAACAGACCT-3′ 725′-CACAGGACAACCAGCTAGC-3′ 73 5′-AATAATCCCATCTCATTGG-3′ 745′-ATTTGAAAAACAGCTAGGCCAAATAA-3′ 75 5′-CTTAGATCCCGTGGTTTACG-3′ 765′-AATCTGTGATGTGCAAAGTATC-3′ 77 5′-AAAGATTCCCTCAGACTCCATTTGT-3′ 785′-CACTTTCACCAACATGGAGG-3′ 120 5′-AGGGTGCATCTTTCCCTTTGTAC-3′ 1215′-GCAGCATGGGACAATACTC-3′ 122 5′-GTGATCAGTCTGCCATTGTG-3′ 1235′-CCTCTGGCAGTAGCCTCGAACATCT-3′ 124 5′-ACTGGTACAAGAAGGGAAGC-3′ 1255′-ACCCACTGATCCAAAGTCC-3′ 126 5′-TCAGCCACTCCTGGCTTTGGG-3′ 1275′-GGCGTCACTTACCTAGCTC-3′ 128 5′-TAGATGTCAGTTTCCTCAGG-3′ 1295′-CCTCCCCCTTTTTCCATTGTGC-3′ 130 5′-TTTCATACGGAGGAGGATGG-3′ 1315′-AGGCTCTACGGCAATAACC-3′ 132 5′-CAACAGTACATCTGCCGGAGAGA-3′ 1335′-GAGAGCCTTATCTAAAAGAGG-3′ 134 5′-CTTTTCCAAACAAGCCACAG 1355′-AACCACTTTTTTCTTGTTCACATCTC 136 5′-TGGAGCTCCGTGTAAAGTC-3′ 1375′-GCACTGTTGGTATTCTCAGC-3′ 138 5′-CCCTTTTTCACACCACAATTACCG-3′ 1395′-GAAAGCCAGATGCTCATTCC-3′ 140 5′-TATCTCTAATTTGCCTAAGTGC-3′ 1415′-CTACCCAGAACAAGCCACTAGC-3′ 142 5′-ATTGTCAATGGGTTTGATAACC-3′ 1435′-TATGATTGAGGCCGCTATCC-3′ 144 5′-TAGTCATGCTTTCAGTCCATTATGC-3′ 1455′-AGTGAACCCAACAACTTTGAC-3′ 146 5′-TGGCTTTTGCCTGCAATCC-3′ Underlinedresidues indicate the positions of the candidate vaccine virus mutationwithin the primer sequence, or, in the case of SEQ ID #52, theunderlined residue indicates the nt-5270 position of DEN-2 16681 virus.

1. A nucleic acid comprising a first nucleic acid molecule encodingnonstructural proteins from an attenuated dengue-2 virus and a secondnucleic acid molecule encoding at least one structural protein from aJapanese encephalitis virus.
 2. The nucleic acid molecule of claim 1,wherein the attenuated dengue-2 virus is vaccine strain PDK-53.
 3. Thenucleic acid of claim 1, wherein the at least one structural proteinfrom the Japanese encephalitis virus is selected from the groupconsisting of a capsid (C) protein, a premembrane (prM) protein, anenvelope (E) protein, and a combination of two or more thereof.
 4. Thenucleic acid of claim 1, further comprising a nucleic acid moleculeencoding a capsid (C) protein from a dengue-2 virus.
 5. The nucleic acidof claim 1, wherein the second nucleic acid molecule encodes a Japaneseencephalitis virus prM protein and a Japanese encephalitis virus Eprotein.
 6. The nucleic acid of claim 5, further comprising a nucleicacid molecule encoding a C protein from a dengue-2 virus.
 7. The nucleicacid of claim 6, wherein the C protein comprises a prM signal sequencefrom a Japanese encephalitis virus.
 8. The nucleic acid of claim 1,wherein the second nucleic acid molecule encodes a Japanese encephalitisvirus C protein and a Japanese encephalitis virus prM protein.
 9. Thenucleic acid of claim 1, wherein the Japanese encephalitis virus is avirulent strain.
 10. A composition comprising at least one nucleic acidof claim 1 and a pharmaceutically acceptable carrier.
 11. Thecomposition of claim 10, further comprising an immunogenic compositionfor a flavivirus selected from the group consisting of a dengue virus, ayellow fever virus, a tick-borne encephalitis virus, a Japaneseencephalitis virus, a West Nile virus, a hepatitis C virus, and acombination of two or more thereof.
 12. A method of inducing an immuneresponse in a subject, comprising administering an effective amount ofthe composition of claim 10 to the subject.
 13. A method of inducing animmune response in a subject, comprising administering an effectiveamount of the composition of claim 12 to the subject.
 14. A nucleic acidcomprising a first nucleic acid molecule encoding nonstructural proteinsfrom an attenuated dengue-2 virus and a second nucleic acid moleculeencoding at least one structural protein from a dengue-2 virus and atleast one structural protein from a Japanese encephalitis virus.
 15. Thenucleic acid of claim 14, wherein the attenuated dengue-2 virus isvaccine strain PDK-53.
 16. The nucleic acid of claim 14, wherein the atleast one structural protein from Japanese encephalitis virus isselected from the group consisting of a prM protein, an E protein, and acombination thereof.
 17. The nucleic acid of claim 14, wherein the atleast one structural protein from dengue-2 virus is a C protein.
 18. Thenucleic acid of claim 17, wherein the C protein comprises a prM signalsequence from a Japanese encephalitis virus.